Biodegradation of phenolic compounds by Basidiomycota and its phenol oxidases: A review.

The phylum Basidiomycota include organisms with enormous bioremediation potential. A variety of processes were proposed at the lab scale for using these fungi and their phenol oxidases in the degradation of phenolics. Here we present a survey of this topic using literature published mostly over the last 10 years. First, the sources of the enzymes are summarized. The laccase and tyrosinase were mainly from Trametes versicolor and Agaricus bisporus, respectively. Recently, however, new promising wild-type producers of the enzymes have emerged and a number of recombinant strains were also constructed, based mainly on yeasts or Aspergillus strains as hosts. The next part of the study summarizes the enzyme and whole-cell applications for the degradation of phenols, polyphenols, cresols, alkylphenols, naphthols, bisphenols and halogenated (bis)phenols in model mixtures or real wastewaters from the food, paper and coal industries, or municipal and hospital sewage. The enzymes were applied as free (crude or purified) enzymes or as enzymes immobilized in various supports or CLEAs, and optionally recycled or used in continuous mode. Alternatively, growing cultures or harvested mycelia were used instead. The products, which were characterized as quinones and their polymers in some cases, could be eliminated by filtration, flocculation or adsorption onto chitosan. The purity of a treated wastewater was monitored using a sensitive aquatic organism. It is concluded that low-cost sources of these enzymes should be searched for and the benefits of enzymatic, biological and physico-chemical methods could be combined to make the processes fit for industrial use.

Biodegradation of brominated aromatics by cultures and laccase of Trametes versicolor.

2-Bromophenol (1), 4-bromophenol (2), 2,4-dibromophenol (3), 2,6-dibromophenol (4), 2,4,6-tribromophenol (5) and tetrabromobisphenol A (6) (1 mM each) added to growing submerged cultures of Trametes versicolor CCBAS 612 were eliminated by 65-85% from the culture medium within 4d. Extracellular laccase activity in the culture medium was influenced by the type of brominated compound added. Maximum level of laccase (63 U L(-1)) was found in the culture with 2-bromophenol. Tetrabromobisphenol A was degraded by a commercial laccase from Trametes versicolor in absence of any oxidation mediator, hydroxylated dibrominated compounds being detected as soluble reaction products by LC/MS. A significant degradation of brominated phenols by laccase was achieved only in the presence of ABTS structural characterization of major products suggesting reaction between bromophenol and ABTS radicals.

Long-term maintenance of fungal cultures on perlite in cryovials - an alternative for agar slants.

Cultures of 33 basidiomycete strains out of 35 tested were viable with unchanged characteristics after four years of maintenance on perlite in cryovials. These cultures can be a good substitute for agar cultures in long-term maintenance of fungi. For comparison, the storage under oil was evaluated but it turned out to be unsuitable for the majority of our cultures.

Cryopreservation of filamentous micromycetes and yeasts using perlite.

The viability, growth and morphology of 48 strains of Ascomycota (including 17 yeasts) and 20 strains of Zygomycota were determined after a 2-d and then after 1-year storage in liquid nitrogen using a new cryopreservation method with perlite as a particulate solid carrier. In case of Ascomycota, 45 strains (94 %) out of 48 survived both 2-d and 1-year storage in liquid nitrogen, respectively. In case of Zygomycota, all 20 strains survived both storage. In addition, 3 strains of Basidiomycota counted among yeasts were tested and all survived the 1 year storage. In all surviving cultures no negative effects of cryopreservation by this method have been observed after 1-year of storage in liquid nitrogen. The results indicate that the perlite protocol can be successfully used for cryopreservation of taxonomically different groups of fungi and also for fungi which failed to survive other routinely used preservation procedures.

Evaluation of synthetic dye decolorization capacity in Ischnoderma resinosum.

The little studied white rot fungus Ischnoderma resinosum was tested for its ability to decolorize seven different synthetic dyes. The strain efficiently decolorized Orange G, Amaranth, Remazol Brilliant Blue R, Cu-phthalocyanin and Poly R-478 on agar plates and in liquid culture at a relatively high concentration of 2-4 and 0.5-1 g l(-1), respectively. Malachite Green and Crystal Violet were decolorized to a lower extent up to the concentration of 0.1 g l(-1). Decolorization capacity of I. resinosum was higher than that in Phanerochaete chrysosporium, Pleurotus ostreatus or Trametes versicolor. In contrast with these thoroughly examined fungi, I. resinosum was able to degrade a wide spectrum of chemically and structurally different synthetic dyes. I. resinosum also efficiently decolorized dye mixtures. In liquid culture, Orange G and Remazol Brilliant Blue R were decolorized most rapidly; the process was not affected by different nitrogen content in the media. Shaken cultivation strongly inhibited the decolorization of Orange G.

Laccase and other ligninolytic enzyme activities of selected strains of Trametes spp. from different localities and substrates.

Eighty-three strains belonging to three species of the genus Trametes FR. (T. versicolor, T. hirsuta and T. ochracea) collected in different localities and on different substrates were screened for laccase production. The production of other lignin-modifying enzymes--manganese peroxidase (MnP) and lignin peroxidase (LiP)--and the decolorization ability were also determined in 21 of them. Production variability was relatively high and no significant correlation was found between the origin of the strains (locality, substrate) and the enzyme production. Dikaryons of all 3 species (but not of all their strains) exhibited LiP activity, which was not detected in the respective monokaryons.

Viability of basidiomycete strains after cryopreservation: comparison of two different freezing protocols.

The viability of 250 basidiomycete strains was determined after a 2-d and then after a 2-year storage under liquid nitrogen using two different freezing protocols. Using an original agar plug protocol (OP), 162 strains (65%) of the 250 strains survived a 2-d storage and 158 strains (63%) survived a 2-year storage in liquid nitrogen. Using a straw protocol (CP), 246 strains (98%) of the 250 strains survived a 2-d storage and 243 strains (97%) a 2-year storage in liquid nitrogen. In addition, other 106 strains were newly estimated using the CP protocol; 104 (98%) of them survived successfully a 2-d storage and 101 (95%) of them survived a 2-year storage in liquid nitrogen. The results indicate that the protocol used for cryopreservation can significantly influence strain survival. Markedly better results were obtained using the CP protocol.

Decolorization of orange G by Pleurotus ostreatus monokaryotic isolates with different laccase activity.

The effect of enhanced laccase (Lac) activity (obtained after copper addition to cultivation media) on decolorization of azo dye Orange G in two basidiospore-derived monokaryotic isolates of Pleurotus ostreatus was determined. The high Lac-producing isolate efficiently decolorized Orange G. The low-producing isolate showed only poor decolorization ability during cultivation in liquid medium and no decolorization on agar plates containing Orange G after a 25-d growth. A substantial enhancement of Lac activity caused by copper addition into cultivation media was detected in both isolates but, at the same time, the biomass production decreased and decolorization rate was reduced.

Decolorization of synthetic dyes by Pleurotus ostreatus isolates differing in ligninolytic properties.

The ability to decolorize four synthetic dyes (Phenol Red, Evans Blue, Eosin Yellowish and Poly B411) in five Pleurotus ostreatus strains (a parental strain and four isolates derived from it) was determined. Two of the isolates had markedly higher and other two substantially lower production of ligninolytic enzymes and hydrogen peroxide than the parental strain. Like the parental strain, the higher-producing isolates were able to decolorize all the tested dyes, but not to a higher extent than the parental strain. In contrast, two lower-producing isolates exhibited slow decolorization, which was incomplete even at the end of cultivation. Evans Blue and Eosin Yellowish strongly suppressed the growth of the strains, while Phenol Red and Poly B411 induced none or only a very slight growth reduction.

Cryopreservation of basidiomycete strains using perlite.

A new alternative method using perlite as a particulate solid carrier in the growth medium with a cryoprotectant was successfully tested for cryopreservation of several basidiomycete species from different genera (Armillaria, Pleurotus, Pluteus, Polyporus) which failed to survive or retain their properties in cryopreservation procedures routinely used in our laboratory. Frozen basidiomycete strains were kept in cryovials submerged in liquid nitrogen and were either immediately after the freezing process or after a 6-month storage thawed and checked for viability, purity and changes in growth, morphology and biochemical characteristics. All cultures survived the cryopreservation procedure and no negative effects of cryopreservation by this method have been observed after 6 months of storage in liquid nitrogen.

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.

Ligninolytic characteristics of Pleurotus ostreatus strain F6 and its monokaryotic protoplast derivative P19.

A stable isolate of Pleurotus ostreatus P19 differing in some morphological and physiological characteristics from its parental wild-type strain F6 was obtained via protoplast isolation during the preparation of strains with altered ligninolytic abilities. The isolate is monokaryotic, does not form clamp-connections, and produces much higher activities of enzymes involved in lignin modification (laccase, manganese peroxidase). Cellulase activity was comparable to that of wild-type strain F6, but the xylanase activity was slightly higher in isolate P19. However, this monokaryotic derivative degrades lignin at a slightly lower rate than its parental strain F6. Electron microscopy observations of wood degradation as a function of mycelium growth were performed on three zones of birch wafers delimited according to the distance from the point of inoculation. The different stages of fungal mycelium growth showed differences in the ultrastructural patterns of the decay not only between the strains P19 and F6, but also depending on the distance from the point of inoculation. This suggests a spatio-temporally controlled secretion of enzymes along the hyphae. The enhanced ability of P19 to degrade the condensed forms of lignin in middle lamellae is correlated to its higher laccase activity.

Screening of Pleurotus ostreatus isolates for their ligninolytic properties during cultivation on natural substrates.

Thirteen basidiospore-derived isolates of Pleurotus ostreatus f6 strain differing in the level of ligninolytic enzyme production and other characteristics (mycelium extension rate, colony morphology) from the parental strain were cultivated on natural substrates. Under these conditions ligninolytic enzyme activity, loss of organic mass, polycyclic aromatic hydrocarbons (PAHs) degradation and colonization of sterile and nonsterile soil were studied. The activity of ligninolytic enzymes was substantially higher in straw than in liquid culture, although the differences between the isolates were less pronounced on this substrate. Some of the isolates showed a very good ability to decompose the lignocellulosic substrate (straw) and a relatively high loss of organic mass was found after 50 days of cultivation in these strains. The original strain f6 and isolates B13 and B26 successfully degraded all seven tested PAH compounds present in experimental soil samples, but the higher or lower ligninolytic enzyme production of isolates tested had no substantial effect on the extent of the degradation. In our screening, six basidiospore-derived isolates growing well in nonsterile soil were found, which could be suitable for the prospective biotechnological exploitation.

Preparation and crossing of basidiospore-derived monokaryons--a useful tool for obtaining laccase and other ligninolytic enzyme higher-producing dikaryotic strains of Pleurotus ostreatus.

Laccase and other ligninolytic enzyme higher-producing dikaryons of Pleurotus ostreatus were obtained after crossing of compatible basidiospore-derived monokaryons selected from the parental basidiospore population on the basis of exceptionality in enzyme production, mycelium extension rate and/or colony morphology. As all detected changes in enzyme activity, mycelium extension rate, colony appearance and degradation of the polymeric dye Poly B411 were relatively stable after repeated testing, the dikaryotic isolates prepared in this way seem to be useful for the future biotechnological exploitation. No correlation between the colony appearance or the mating type and the enzyme activity or other characteristics tested has been found.

Activities of ligninolytic enzymes in some white-rot basidiomycete strains after recovering from cryopreservation in liquid nitrogen.

Fourteen strains of white-rot basidiomycetes belonging to eight species of two genera (Inonotus and Pholiota) were tested for their ability to maintain the production of laccase, peroxidase and manganese-dependent peroxidase (enzymes involved in lignin biodegradation) after a short-time preservation in liquid nitrogen with different cryoprotectives (glycerol, dimethyl sulfoxide). No negative effect of cryopreservation or the used cryoprotective on production of the ligninolytic enzymes was found in the fungi tested.

Degradation of wood by the basidiomycete Coriolopsis occidentalis.

Chemical and microscopic features of wood decay by the basidiomycete Coriolopsis occidentalis are described. The fungus was grown on blocks of poplar, oak, and fir wood and caused significant mass, lignin, and saccharide losses in all kinds of wood. Poplar wood was particularly strongly affected. Twelve weeks after inoculation dry mass, lignin, and saccharide contents were reduced by about 50%. The blocks became covered with mycelia and electron microscopy showed that secondary cell walls were degraded from the lumina and middle lamellae dissolved during later stages of incubation. The results indicate that the fungus belongs to simultaneous white-rotters.

Genetic Mapping in the Lignin-Degrading Basidiomycete Phanerochaete chrysosporium.

A method of meiotic segregation analysis based on recombinant selection in the homothallic basidiomycete Phanerochaete chrysosporium was developed. Using this method, we were able to reveal linkage relationships and to estimate recombination frequencies between seven mutations to auxotrophy. We detected two linkage groups, the first containing four and the second three of the seven mapped mutations.

In vitro adrenocorticotropin/beta-endorphin-releasing activity of vasopressin analogs is related neither to pressor nor to antidiuretic activity.

The ability of vasopressin and related analogs to induce ACTH, beta-endorphin, and beta-lipotropin release was studied in vitro using incubated rat anterior pituitary quarters or a perifused rat isolated anterior pituitary cell column. Vasopressin and its analogs exhibited corticotropin-releasing factor (CRF)-like activity in a rank order which was different from those for vasopressor or antidiuretic activity. Two dissimilar antagonists with antivasopressor activity showed different effects: one possessed CRF-like activity itself, the other blocked the CRF-like activity of vasopressin. Another antagonist with antipressor and also antiantidiuretic activity had no effect when given alone and also didn't block the CRF-like activity of vasopressin. Some analogs were also tested for their effects on cAMP accumulation. Analogs, which possessed CRF-like activity or blocked CRF-like activity of vasopressin, stimulated cAMP accumulation or inhibited vasopressin-stimulated cAMP accumulation in anterior pituitary quarters, respectively. These results imply that the structural requirements of the CRF-like activity of vasopressin differ from those of the pressor and antidiuretic activity. Therefore, it is possible that the pituitary receptors responsible for CRF-like activity of vasopressin represent a separate category of vasopressin receptors which may be linked to an adenylate cyclase.

Stimulation of adrenocorticotropin/beta-endorphin release by synthetic ovine corticotropin-releasing factor in vitro. Enhancement by various vasopressin analogs.

Vasopressin analogs, which markedly differed in the ratio of pressor versus antidiuretic activity and also in ACTH/beta-endorphin-releasing activity, were used in the present study. The ability of vasopressin and these analogs to enhance the release of adrenocorticotropin-(ACTH-IR) and beta-endorphin-like immunoreactivity (beta-EI) induced by synthetic ovine corticotropin-releasing factor -CRF-(1-41)- was studied in vitro using incubated rat anterior pituitary quarters. Arginine vasopressin (AVP) potentiated the action of CRF-(1-41) 2- to 4-fold. Vasopressin analogs, which possess direct CRF-like activity when given alone, also enhanced beta-EI release caused by CRF-(1-41); the lowest concentration able to potentiate CRF-(1-41) activity was closely correlated with the ED50 value of these analogs for direct CRF-like activity. The vasopressin analog 1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid)-2-(O-methyl)tyrosine-8-arginine-vasopressin blocked the release of ACTH-IR induced by AVP; however, this analog failed to prevent the potentiation by AVP of ACTH-IR release caused by CRF-(1-41), but enhanced itself the action of CRF-(1-41). Two analogs, which exhibited no direct CRF-like activity and which also did not antagonize the CRF-like activity of AVP, markedly enhanced the ACTH-IR and beta-EI response to CRF-(1-41).(ABSTRACT TRUNCATED AT 250 WORDS)