Screening for dioxin-degrading basidiomycetes from temperate and tropical forests

A total of 500 strains of basidiomycetes isolated from temperate forests in Japan and 379 strains from tropical forests in Indonesia were subjected to a laboratory screening for dioxin-degrading ability. At first, about 200 fungal strains were selected by their ability to decolorize Remazol Brilliant Blue R dye as an indicator of ligninolytic activities. Next, for excluding the factor of dioxin sorption by mycelia, we prepared two series of living cultures exposed either long-term or short-term to 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD), and compared the decreases in the remaining amounts of this model compound. We chose Bjerkandera adusta strain VH57 as a promising new candidate for dioxin degradation, because it gave 40% difference in 2,7-DCDD levels between the two treatments after 30 days of exposure.     

Models of 2,4,6-trinitrotoluene (TNT) initial conversion by yeasts

We devised a screening method to obtain basidiomycetous fungi capable of degrading dioxins. About 200 fungal strains were selected from more than 1500 strains by their ability to decolorize Remazol brilliant blue R dye as an indicator. To attempt to eliminate the factor of dioxin sorption by mycelia, we prepared two series of living cultures exposed either long term or short term to 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD), and compared the decreases in the levels of this chemical. In only 11 strains was there a significant difference between the two treatments. We chose Panellus stypticus strain 99-334 as a new, effective dioxin degrader, because it gave a close to 100% decrease in 2,7-DCDD levels (from an initial concentration of 10 microM) after 40 days of exposure. The detection of a metabolic intermediate (1-chloro-3,4-dihydroxybenzene) by gas chromatography-mass spectrometry analysis supported the ability of this strain to degrade 2,7-DCDD.     

Biotransformation of acetamiprid by the white-rot fungus Phanerochaete sordida YK-624

Acetamiprid (ACE) belongs to the neonicotinoid class of systemic broad-spectrum insecticides, which are the most highly effective and largest-selling insecticides worldwide for crop protection. As neonicotinoid insecticides persist in crops, biotransformation of these insecticides represents a promising approach for improving the safety of foods. Here, the elimination of ACE from a liquid medium by the white-rot fungus Phanerochaete sordida YK-624 was examined. Under ligninolytic and non-ligninolytic conditions, 45% and 30% of ACE were eliminated, respectively, after 15 days of incubation. High-resolution electrospray ionization mass spectra and nuclear magnetic resonance analyses of a metabolite identified in the culture supernatant suggested that ACE was N-demethylated to (E)-N ¹-[(6-chloro-3-pyridyl)-methyl]-N ²-cyano-acetamidine, which has a much lower toxicity than ACE. In addition, we investigated the effect of the cytochrome P450 inhibitor piperonyl butoxide (PB) on the elimination of ACE. The elimination rate of ACE by P. sordida YK-624 was markedly reduced by the addition of either 0.01 or 0.1 mM PB to the culture medium. These results suggest that cytochrome P450 plays an important role in the N-demethylation of ACE by P. sordida YK-624.     

Biotransformation of polychlorinated dibenzo-<Emphasis Type="Italic">p</Emphasis>-dioxin by <Emphasis Type="Italic">Coprinellus</Emphasis> species

A degradation experiment on dibenzo-p-dioxin (DD) and 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD) was carried out using basidiomycetous fungi belonging to the genera Coprinus, Coprinellus, and Coprinopsis. Some species showed a high rate of decrease in DD for the 2-week test period. Among them, Coprinellus disseminatus showed the highest ability to decrease the DD level, close to 100% by the end of 2 weeks. Further examination showed that Coprinellus disseminatus and Coprinellus micaceus, belonging to the genus Coprinellus, were able to metabolize 2,7-DCDD to a monohydroxylated compound, probably mediated by the P450 system. The metabolism of chlorinated DD by fungi capable of living in soil conditions is reported here for the first time.     

Transformation by complementation of a uracil auxotroph of the hyper lignin-degrading basidiomycete Phanerochaete sordida YK-624

Phanerochaete sordida YK-624 is a hyper lignin-degrading basidiomycete possessing greater ligninolytic selectivity than either P. chrysosporium or Trametes versicolor. To construct a gene transformation system for P. sordida YK-624, uracil auxotrophic mutants were generated using a combination of ultraviolet (UV) radiation and 5-fluoroorotate resistance as a selection scheme. An uracil auxotrophic strain (UV-64) was transformed into a uracil prototroph using the marker plasmid pPsURA5 containing the orotate phosphoribosyltransferase gene from P. sordida YK-624. This system generated approximately 50 stable transformants using 2 × 10⁷ protoplasts. Southern blot analysis demonstrated that the transformed pPsURA5 was ectopically integrated into the chromosomal DNA of all transformants. The enhanced green fluorescent protein (EGFP) gene was also introduced into UV-64. The transformed EGFP was expressed in the co-transformants driven by P. sordida glyceraldehyde-3-phosphate dehydrogenase gene promoter and terminator regions.     

Biodegradation of diphenyl ethers by a copper-resistant mutant ofErwinia sp.

Summary A bacterium tentatively identified as anErwinia sp. was isolated from sewage by enrichment on methanol and lignin. Several mutants developed from this strain were studied for their ability to degrade aromatic ethers. Different concentrations of the chemicals were incubated with the organisms and the degradation was estimated by high-performance liquid chromatography (HPLC). Among these mutants, one isolate,Erwinia sp. strain CU3614, showed resistance to copper ions (>20 mM CuSO₄) and the ability to degrade 4-hydroxydiphenyl ether (4-HDPE), 4-chlorodiphenyl ether (4-CDPE), 4-nitrodiphenyl ether (4-NDPE) and 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD) in the presence of copper ions. Increased concentrations of copper in the medium resulted in higher degradation of 4-HDPE. Further studies with copper-sensitive mutants obtained fromErwinia sp. CU3614 by Tn5 transposon-induced mutagenesis showed a corresponding decrease in the ability to degrade 4-HDPE. These results suggest the presence of a copper-associated activity in the biotransformation of aromatic ethers.     

Improvement of ligninolytic properties by recombinant expression of glyoxal oxidase gene in hyper lignin-degrading fungus Phanerochaete sordida YK-624

Glyoxal oxidase (GLOX) is a source of the extracellular H₂O₂ required for the oxidation reactions catalyzed by the ligninolytic peroxidases. In the present study, the GLOX-encoding gene (glx) of Phanerochaete chrysosporium was cloned, and bee2 promoter of P. sordida YK-624 was used to drive the expression of glx. The expression plasmid was transformed into a P. sordida YK-624 uracil auxotrophic mutant (strain UV-64), and 16 clones were obtained as GLOX-introducing transformants. These transformants showed higher GLOX activities than wild-type P. sordida YK-624 and control transformants harboring marker plasmid. RT-PCR analysis indicated that the increased GLOX activity was associated with elevated recombinant glx expression. Moreover, these transformants showed higher ligninolytic activity than control transformants. These results suggest that the ligninolytic properties of white-rot fungi can be improved by recombinant expression of glx.     

Removal of diclofenac and mefenamic acid by the white rot fungus <Emphasis Type="Italic">Phanerochaete sordida</Emphasis> YK-624 and identification of their metabolites after fungal transformation

The non-steroidal anti-inflammatory drugs diclofenac (DCF) and mefenamic acid (MFA) were treated with the white rot fungus Phanerochaete sordida YK-624. DCF completely disappeared and MFA decreased by about 90% after 6 days of treatment. It was also confirmed that the fungus almost completely removed the acute lethal toxicity of DCF and MFA towards the freshwater crustacean Thamnocephalus platyurus after 6 days of treatment. Mass spectrometric and ¹H nuclear magnetic resonance analyses demonstrated that two mono-hydroxylated DCFs (4′-hydroxydiclofenac and 5-hydroxydiclofenac) and one di-hydroxylated DCF (4′,5-dihydroxydiclofenac) were formed via fungal transformation. The four metabolites of MFA were identified as 3′-hydroxymethylmefenamic acid (mono-hydroxylated MFA), 3′-hydroxymethyl-5-hydroxymefenamic acid (di-hydroxylated MFA), 3′-hydroxymethyl-6′-hydroxymefenamic acid (di-hydroxylated MFA) and 3′-carboxymefenamic acid. These results suggest that hydroxylation catalyzed by cytochrome P450 (CYP) in P. sordida YK-624 may be involved in the elimination and detoxification of DCF and MFA. This notion was further supported by the fact that smaller decreases in DCF and MFA were observed in cultures of P. sordida YK-624 incubated with 1-aminobenzotriazole, a known inhibitor of CYP.     

Lignin peroxidase is involved in the biobleaching of manganese-less oxygen-delignified hardwood kraft pulp by white-rot fungi in the solid-fermentation system

Biobleaching of manganese-less oxygen-delignified hardwood kraft pulp (E-OKP) by the white-rot fungi Phanerochaete sordida YK-624 and P. chrysosporium was examined in the solid-state fermentation system. P. sordida YK-624 possessed a higher brightening activity than P. chrysosporium, increasing pulp brightness by 13.4 points after seven days of treatment. In these fermentation systems, lignin peroxidase (LiP) activity was detected as the principle ligninolytic enzyme, and manganese peroxidase and laccase activities were scarcely detected over the course of treatment of E-OKP by either fungus. Moreover, a linear relationship between brightness increase and cumulative LiP activity was observed under all tested culture conditions with P. sordida YK-624 and P. chrysosporium. These results indicated that LiP is involved in the brightening of E-OKP by both white-rot fungi.     

Trapping of 2,7-Dichlorodibenzo-<Emphasis Type="Italic">p</Emphasis>-Dioxin in Aqueous Solution by Enzymatic Reaction of Fungal Manganese Peroxidase in the Presence of Polyunsaturated Fatty Acids

Abstract:In the presence of polyunsaturated fatty acids, including cis-4,7,10,13,16,19-docosahexaenoic acid (DHA), 2,7-dichlorodibenzo-p-dioxin (DCDD) was treated with manganese peroxidase (MnP) from white rot basidiomycete Phanerochaete sordida YK-624. After incubation with MnP, DCDD could not be extracted from the reaction mixture with n-hexane and was trapped in the water layer. DCDD was released by alkalification of the water layer. DCDD was also trapped after treatment with lipoxidase, which produces hydroperoxides from unsaturated lipids. The amounts of thiobarbituric acid-reactive substances produced in the MnP reactions with three highly unsaturated fatty acids were higher than the amounts produced with three fatty acids with a lower degree of unsaturation. These results suggest that a DCDD-trapping compound may be produced by peroxidation of the polyunsaturated fatty acids.Received: 22 October 2002 / Accepted: 6 December 2002     

Importance of Nutrient Competition and Allelopathic Effects in Suppression of the Green Alga Scenedesmus obliquus by the Macrophytes Chara, Elodea and Myriophyllum

Possible allelopathic effects of substances released from the macrophytes Chara globularis, Elodea canadensis, Myriophyllum spicatum on the common green alga Scenedesmus obliquus were tested in the laboratory with plastic plants and untreated medium as controls. A two-phase approach was used in which first the effects of physical presence of plants was studied (phase I) followed by the effects of plant culture filtrates (phase II). In the presence of plastic plants growth was reduced only marginally, but strong growth inhibition of Scenedesmus occurred in the physical presence of all macrophytes. In contrast, filtrates from Chara had no growth inhibitory effect on Scenedesmus. Myriophyllum filtrate reduced particle-based growth rate by 7% compared to filtration controls, while Elodea culture filtrate reduced volume-based growth by 12%, chlorophyll-based growth by 28% and particle-based growth by 15%. Photosystem II-efficiency of Scenedesmus was reduced in all three macrophyte treatments in phase I, but not in filtrates from macrophyte cultures (phase II). Thus, while enzyme activity or other physiological aspects may have been affected, the current study yielded no proof for allelopathically active compounds being directed at photosynthesis. Mean particle volume (MPV) of Scenedesmus was not influenced by macrophyte exudates and cultures remained dominated by unicells. The strong growth inhibitory effects found for Scenedesmus in the physical presence of macrophytes, but not in plastic controls, and no or weaker response in nutrient-enriched filtrates, suggest nutrient competition was a more powerful driving factor than allelochemicals. However, the experimental design does not exclude disappearance of allelochemicals during the filtration process.     

Immunochemical detection of ochratoxin A in black Aspergillus strains

One hundred and fifty-seven strains belonging to Aspergillus section Nigri were tested for ochratoxin A production using three different methods: a relatively new immunochemical method based on an enzyme-linked immunosorbent assay (ELISA), thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The monoclonal antibody-based ELISA technique was successfully used to screen for low levels of ochratoxin A in the black Aspergilli without concentrating the culture filtrates. The results were confirmed by TLC and HPLC analysis and chemical derivatization. These latter methods required concentrated filtrates. Ochratoxin A was detected in the culture filtrates of five of the 12 A. carbonarius strains, none of the 45 A.japonicus strains and three of the 100 isolates in the A. niger aggregate (A. foetidus, A. awamori and A. niger).Abbreviations ELISA enzyme-linked immunosorbent assay - HPLC high-performance liquid chromatography - OA ochratoxin A - TLC thin-layer chromatography     

Phylogenetical approach to isolation of white-rot fungi capable of degrading polychlorinated dibenzo-p-dioxin

A degradation experiment on PCDDs and phylogenetical analyses were carried out on newly isolated 2,7-dichlorodibenzo-p-dioxin (2,7-diCDD)-degrading white-rot fungi, strains BMC3014, BMC9152, and BMC9160. When these fungi were incubated with tri- or tetraCDDs, the substrates were degraded efficiently, and hydroxylated metabolites were detected. On the other hand, 1,3,6,8-tetrachlorodibenzo-p-dioxin was not decreased, and no metabolites were detected. Phylogenetic analysis of internal transcribed spacers (ITSs) containing rRNA gene sequence (ITS-rDNA) clarified that these strains belonged to the genus Phlebia and were closely related to the fungi Phlebia lindtneri, strains MZ-227 and MG-60, which had both been isolated as 2,7-diCDD-degrading fungi in our previous study. Based on this phylogenetical relationship, other Phlebia genera species were used for a degradation experiment on 2,7-diCDD and 1,3,6,8-tetraCDD. Phlebia acerina and Phlebia brevispora degraded 2,7-diCDD about 40 and 80%, respectively, over 14 days of incubation. It became clear that P. brevispora can degrade 1,3,6,8-tetraCDD and transform it to monohydroxy-tetraCDD, monomethoxy-tetraCDD, dimethoxy-tetraCDD, dimethoxy-triCDD, and 3,5-dichlorocatechol in the treatment cultures. In this paper, we could clearly prove for the first time by identifying the metabolites that white-rot fungus P. brevispora could degrade the recalcitrant dioxin, 1,3,6,8-tetraCDD.     

Production and partial characterization of extracellular proteinases from <Emphasis Type="Italic">Streptomyces malaysiensis</Emphasis>, isolated from a Brazilian cerrado soil

Streptomyces malaysiensis AMT-3, isolated from a Brazilian cerrado soil, showed proteolytic activities detected by gelatin–sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum proteinase production was obtained when using 2.5% wheat bran and 0.1% yeast extract in the culture medium, after 5 days incubation at 30°C. The enzymatic complex degraded gelatin optimally at pH 7.0, and under these conditions eight proteolytic bands (four serine-proteinases and four metaloproteinases), ranging from 20 to 212 kDa, were detected on the culture supernatant filtrates. In addition, a 35-kDa proteinase was thermostable at 60°C for 120 min. These results point out to the applicability of gelatin zymograms in the characterization of crude enzymatic complexes. According to our results, this enzymatic complex could be used for biotechnological applications.     

In Vitro selection of groundnut cell lines fromCercosporidium personatum culture filtrates and regeneration of resistant plants through cell culture

Cell suspensions derived from immature leaves of the groundnut (Arachis hypogaea L.) were cultured in the presence and absence ofCercosporidium personatum pathotoxic culture filtrates. Cell viability and reactions of cell lines were determined after exposure to various concentrations (25–100%, v/v) of the filtrates. Cell lines have been selected for resistance to the toxin(s) produced byC. personatum. Selected cell lines were used for plant regeneration on regeneration media containingC. personatum culture filtrates. Plant regeneration frequency was found to be low in long-term cultures, whereas it was high in short-term cultures. The selfed progeny of the plants regenerated from the resistant cell lines showed resistance to the pathogen in the field. Six out of 82 plants exhibited enhanced resistance in the R₂ generation. The culture filtrate stimulated callus proliferation as well as plant regeneration at lower concentrations, a response that could prove to be very useful for obtaining disease resistant plants throughin vitro selection.     

Allelopathic effects of the submerged macrophyte <Emphasis Type="Italic">Potamogeton malaianus</Emphasis> on <Emphasis Type="Italic">Scenedesmus obliquus</Emphasis>

Allelopathic effects of the submerged macrophyte Potamogeton malaianus on Scenedesmus obliquus were assessed using a two-phase approach under controlled laboratory conditions. In the co-culture experiment (phase І), the growth of S. obliquus at two different initial cell densities was significantly inhibited by P. malaianus. Moreover, the growth inhibition was dependent on the biomass density of P. malaianus. Antioxidant enzymes (SOD, CAT and POD), MDA, APA, total soluble protein, protein electrophoretic pattern and morphology of S. obliquus were determined after the co-culture experiment was terminated. The activities of SOD, CAT, POD and APA at the low initial cell density were stimulated, the contents of MDA and total soluble protein were increased, and some special protein bands disappeared in P. malaianus treatments. The macrophyte had no effect on the activities of SOD and APA at the high initial cell density, but significantly influenced other physiological parameters of S. obliquus with the increase of biomass density. The morphology of S. obliquus showed no difference in the macrophyte treatments and the controls, and the cultures were dominated by 4-celled coenobia. The results indicated P. malaianus had significant allelopathic effects on the growth and physiological processes of S. obliquus. Moreover, the allelopathic effects depended on initial algal cell density, biomass density of the macrophyte, and their interaction. In the experiment of P. malaianus culture filtrates (phase II), filtrates from combined culture of plant and S. obliquus at the low initial cell density exhibited no apparent growth inhibitory effect on S. obliquus. The result showed that initial addition of growth-inhibiting plant filtrates had no allelopathic effect on S. obliquus. We concluded that the allelopathic effects on S. obliquus were found in the presence of P. malaianus, but not in P. malaianus filtrates. However, the absence of allelopathic effect on S. obliquus might be due to the very low concentrations of allelochemicals in the filtrates. Handling editor: S. M. Thomas     

Trapping of 2,7-dichlorodibenzo-p-dioxin in aqueous solution by enzymatic reaction of fungal manganese peroxidase in the presence of polyunsaturated fatty acids

In the presence of polyunsaturated fatty acids, including cis-4,7,10,13,16,19-docosahexaenoic acid (DHA), 2,7-dichlorodibenzo-p-dioxin (DCDD) was treated with manganese peroxidase (MnP) from white rot basidiomycete Phanerochaete sordida YK-624. After incubation with MnP, DCDD could not be extracted from the reaction mixture with n-hexane and was trapped in the water layer. DCDD was released by alkalification of the water layer. DCDD was also trapped after treatment with lipoxidase, which produces hydroperoxides from unsaturated lipids. The amounts of thiobarbituric acid-reactive substances produced in the MnP reactions with three highly unsaturated fatty acids were higher than the amounts produced with three fatty acids with a lower degree of unsaturation. These results suggest that a DCDD-trapping compound may be produced by peroxidation of the polyunsaturated fatty acids.     

Selection for resistance to filtrates of Fusarium spp. in embryogenic cell suspension culture of Medicago sativa L.

A highly embryogenic cell suspension of alfalfa derived from a genotype sensitive to Fusarium oxysporum was successfully used for selection in vitro for resistance to culture filtrates of F. oxysporum, F. solani and F. avenaceum. Fifty two stable resistant cell lines were obtained and 500 plants regenerated from them. Among the 167 regenerants tested under glass there were 12–20% more plants with increased resistance to pathogens than in the group of plants regenerated from a control cell line. It was also found that the cell suspension cultures derived from genotypes of alfalfa with increased resistance to Fusarium spp. better tolerated filtrates of the pathogen. The results of a comparison of virulence of individual isolates of several species of Fusarium with toxicity of their filtrates to plants in vivo and in cell cultures were not unequivocal.     

Influence of Fusarium wilt toxin(s) on carnation cells

The influence of culture filtrates of Fusarium oxysporum f.sp. dianthi which causes Fusarium wilt was investigated on growth and viability of carnation tissue cultures and leaf segments. Culture filtrates of avirulent race 1 of this fungus did not affect calli and leaf segments of cultivars both susceptible and resistant to Fusarium wilt. However, culture filtrates of virulent race 2 decreased viability and suppressed growth of callus of the susceptible cultivar. In contrast, callus of the resistant cultivar showed resistance to the culture filtrates. The results of these experiments may provide information on methods of selection of new wilt resistant carnation varieties.Abbreviations A270 absorbance at 270 nm - 2,4-d 2,4-dichlorophenoxyacetic acid - CF-MCD culture filtrate of 16064 grown in MCD medium - MCD medium modified Czapeck-Dox medium - MS medium basal medium of Murashige and Skoog - MW molecular weight - PD medium potato dextrose medium - TTC 2,3,5-triphenyl tetrazolium chloride     

Studies on mineral phosphate solubilization by cyanobacteria <Emphasis Type="Italic">Westiellopsis</Emphasis> and <Emphasis Type="Italic">Anabaena</Emphasis>

Two diazotrophic cyanobacteria, Westiellopsis prolifica and Anabaena variabilis were evaluated for elucidating the possible mechanism of mineral phosphate solubilization. Phosphate starved cyanobacteria evaluated for the presence of organic acids, extracellular compounds or enzymes that might have been produced and promoted the mineral phosphate solubilization with Mussorie Rock Phosphate and Tricalcium Phosphate as substrates. Both the cultures did not reveal production of organic acids throughout the incubation period when checked for decrease in media pH of the media and thin layer chromatography. Thin layer chromatography of culture filtrates showed the presence of hydrocarbon like compound. Further analysis of the culture filtrates with gas liquid chromatography, a single peak near to the retention time of 7.6 was observed in all extracts of culture filtrates irrespective of phosphate source. UV-visible spectra of culture filtrates revealed the absorption maxima of 276 nm. Gas chromatographic-mass spectrometric analysis of culture filtrates showed most intense peak in the electron impact (EI) ionization was at m/z 149 and molecular ion peaks at m/z 207 and 167, inferring the presence of phthalic acid. Among the mechanisms in mineral phosphate solubilization, it was evident that these cyanobacteria used phthalic acid as possible mode of P solubilization.