Hans-Peter Molitoris

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.

Scanning electron microscopy of polyhydroxyalkanoate degradation by bacteria

Bacterial degradation of sheets of selected polyhydroxyalkanoates by Comamonas sp., Pseudomonas lemoignei and Pseudomonas fluorescens GK13 is reported. Five natural polyhydroxyalkanoates were used, namely poly(3-hydroxybutyrate), poly(3-hydroxyvalerate), a copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate, a copolymer of mainly 3-hydroxyoctanoate and minor amounts of 3-hydroxyhexanoate, and two rubber-like copolymers of saturated and unsaturated hydroxyalkanoic acids that had been modified by electron-beam-induced cross-linking. Each of these polymers was degraded by at least one bacterial strain, the rate of hydrolysis being dependent on the surface area of the polymer exposed to attack. Scanning electron microscopy of partially degraded samples showed that hydrolysis started at the surface and at physical lesions in the polymer and proceeded to the inner part of the material. No evidence for areas of non-degradable polymer was found for any of the polymers analysed, even if the polymer contained chemical cross-links.

Mushrooms in Medicine

Fungi have played an important role as food, medicine, poison and for religious and other purposes in the life of man since prehistoric times. The role in medicine of higher (macro-)fungi in different countries from early historic times through the Middle Ages until now and also their prospective use in the future is described. Significant changes in the use of fungi for medical purposes are shown and some current and future trends are exemplified. Throughout the review, the role of Czechoslovak scientists in this field, starting from taxonomy and ending in the production of beneficial drugs from fungi is indicated.

Cultivation of fungi under simulated deep sea conditions

In order to investigate the barotolerance of marine fungi and to elucidate their ecological role in the deep sea, high-pressure equipment was built and tested. Cultures of the marine yeasts Debaryomyces hansenii, Rhodotorula rubra and Rhodosporidium sphaerocarpum were inoculated into gas-permeable plastic foil bags and incubated in pressure vessels filled with hydraulic fluid that serves also as an oxygen reservoir. The equipment was used at temperatures from 7° to 34°C and pressures from 0.1 to 80.0 MPa. Comparison of five types of plastic foil showed effects on total cell number in batch culture. An exchange of the hydraulic fluid increased yield by replenishing oxygen, the growth-limiting factor. There were no detectable growth differences between buffered (TRIS, HEPES, imidazole, MES) and unbuffered media and thus unbuffered medium was used. Yeasts were best cultivated in an unbuffered sea-water medium (glucose—peptone—yeast extract) sealed in bags made from polyethylene foil. The fluorocarbon liquid FC-77 provided the best oxygen reservoir owing to its high oxygen and carbon dioxide solubility. An exchange of the hydraulic fluid is not necessary if sensitive growth determination methods are used. All marine yeasts cultivated under simulated deep sea conditions were able to grow at least up to a pressure of 20 MPa, with Rhodotorula rubra and Rhodosporidium sphaerocarpum growing at 40 MPa, corresponding to 4000 m depth. Thus, marine yeasts are able to grow under simulated deep sea conditions and may participate in the degradation of organic matter in the deep sea.

Comparative synthesis and hydrolytic degradation of poly (L-malate) by myxomycetes and fungi

Various fungi and myxomycetes have been tested for their synthesis and hydrolytic degradation of β-poly(L-malate) (PMLA), a highly anionic polyester of L-malate. The PMLA content of the culture medium, and also of some cell extracts, has been assayed by L-malate dehydrogenase (EC 1.1.1. 83)-catalysed reduction of NAD + after the alkaline hydrolysis of the polymer. All eight myxomycete isolates were producers. Eight producers were Mitosporic fungi but only one Ascomyete was productive. Aureobasidium pullulans contained PMLA in yeast-like cells but not in hyphae. The polymer was bound to membrane fractions from where it was slowly released during preparation for analysis. Only 12 of the 237 fungi and myxomycetes showed PMLA-hydrolase activity; and two of these were producers of PMLA. Fungi with hydrolase activity were mostly different from those releasing L-malate. The Physarum polycephalum -type unbranched polymer of high molecular mass was not recognized in fungi. Fungi, typically A. pullulans , produced polymer of low molecular mass which seemed to be covalently bound to polysaccharide.

Biological decolorization of the synthetic dye RBBR in contaminated soil

Soil contaminated with the synthetic dye Remazol Brilliant Blue R (RBBR) was treated independently with the wheat straw-grown white rot fungus Irpex lacteus, a bacterial consortium isolated from a dye-polluted soil and a coculture comprising both I. lacteus and the bacterial consortium. Both I. lacteus and the coculture removed RBBR (decrease in absorbance at 578 nm) gradually during a 49-day incubation time to 76 and 78%, respectively. The bacterial consortium alone, however, decolorized RBBR starting after 14 days with a final RBBR removal of 89%. Using controls with heat-killed cultures almost no decolorization occurred. The decolorization by the coculture did not show an increased RBBR removal as compared to the individual cultures. This might be explained by the observation that I. lacteus inhibited growth of the bacterial consortium.

The phenoloxidases of the ascomycete Podospora anserina X. Electron microscopic studies on the structure of laccases I, II and III

Electron microscopy of three intracellular laccases from the Ascomycete Podospora anserina showed that the high molecular weight laccase I (Mr = 390 000) appears mainly as tetramers with the four subunits arranged at the corners of rectangles (13.3 nm × 8.5 nm). In size and shape the subunits of laccase I equal the monomeric molecules of laccase II (Mr = 70 000, 6.8 nm × 5.5 nm) and laccase III (Mr = 80 000, 7.0 nm × 5.8 nm). The dimensions of the monomeric laccases estimated from the electron micrographs are in good agreement with the hydrodynamic measurements of their molecular weight.

Pilze in Medizin, Folklore und Religion

Pilze spielen in der Menschheitsgeschichte, in Folklore, Legenden und Religion, eine wichtige Rolle als Nahrung, Sucht- und Heilmittel. Nach einer kurzen Einfuhrung in die Natur der Pilze, den faszinierenden Lebewesen zwischen Pflanze und Tier, wird ein historischer und medizinischer Uberblick gegeben, beginnend mit den fruhesten Nachweisen und erganzt durch Abbildungen aus alten und neuen Quellen. Wichtige Beispiele werden veranschaulicht durch Hinweise auf ihre Rolle als Nahrung oder Gift, in Medizin, Folklore und Religion, beginnend in der Vergangenheit, uber die Gegenwart zur Zukunft. In der Vergangenheit wurden bis in das Mittelalter Pilze in der ostasiatischen und in der westlichen Medizin – neben ihrer Bedeutung als Nahrungsmittel – vorwiegend frisch oder getrocknet als “Droge” eingesetzt, wie dies in Text und Bild aus den beruhmten Krauterbuchern der “Vater der Botanik” hervorgeht. Zusatzlich wurden sie entsprechend der Medizintheorie der “Signaturlehre” als Heilmittel nach ihrer Form und Farbe verwendet. Die gegenwartige Bedeutung von Pilzen in der Medizin wird anhand einiger Beispiele erlautert, wobei die fruhere “Droge” durch aus den Pilzen gereinigte, aktive Substanzen ersetzt wird, die zunehmend biotechnologisch produziert oder sogar (bio)synthetisch hergestellt werden. Nach wie vor sind Pilze aber noch lebendig in Folklore und Legende als “Hexeneier”, “Hexenringe”, in Kinderbuchern und als Glucksbringer. Fur die Zukunft wird gezeigt, dass Pilze auch hier eine zunehmende Bedeutung zu erwarten haben, insbesondere in Medizin und Biotechnologie aufgrund ihrer Stoffwechseleigenschaften und Syntheseprodukte. Sie werden also in Medizin, Legenden und Folklore uberleben und uns dabei an langst vergangene Zeiten erinnern. Mushrooms in medicine, folklore and religion Mushrooms have played an important role in human history as food, as poison, as medicine, in folklore, legends and religion. All these areas are intimately interrelated. After a short introduction into the nature of mushrooms or “Toadstools”, representing fascinating organisms between the kingdom of plants and animals, a historical and medical review is given, supplemented by pictures from old and new origines, starting with the earliest sources where mushrooms appeared. Whenever possible, pictures or names of mushrooms are followed by anecdotes on their role as food or poison, in medicine, folklore and religion. This starts with mushrooms in the past, goes on to the present and leads to their possible significance in the future. In early history, until medieval times, mushrooms – beside being used as food – were used in medicine primarily as fresh or dried “drug”, as it is shown in the famous herbals of the “fathers of botany”, such as Fuchs and Matthiolus in Germany or Turner or Gerard in England, where mushrooms are described, pictured and their uses in medicine and folklore evaluated. In addition, mushrooms were used in these times as medicaments also just by their form and colour following the “doctrine of signatures”. The present significance of mushrooms and their applications in medicine are given by a number of examples, where the former “drug” is replaced by the purified active substances, which increasingly are produced biotechnologically or even are biosynthesized. Still mushrooms are lively in folklore as “witches eggs”, “fairy rings”, fortune bringers or in other connotations such as children rhymes. Finally, it will be shown that mushrooms also in the future are expected to have an increasing importance, particularily in medicine and biotechnology by their unique biosynthetic capabilities and metabolic products. They therefore should survive also in legends, myth and folklore, often reminding us to long bygone times.

Screening of fungal strains for remediation of water and soil contaminated with synthetic dyes

Using Remazol Brilliant Blue R (RBBR), strains of terrestrial white rot (WRF) and marine fungi (MF) were screened for efficient decolorization. Dye degradation potential of selected strains was studied with chemically different dyes (azo, anthraquinone, heterocyclic, triphenylmethane). Irpex lacteus and Pleurotus ostreatus (WRF) efficiently degraded dyes from all groups whereas less efficient and selective degradations were observed with Dactylospora haliotrepha and Aspergillus ustus (MF). Seawater salinity often reduced decolorization efficiency of WRF but increased decolorization ability of MF. In soil I. lacteus removed 77% of RBBR used at 150 μg/g within 6 weeks. The work presents fungi as suitable candidates to be applied to re-mediation of dye-contaminated water and soil.

THE PHENOLOXIDASES OF THE ASCOMYCETE PODOSPORA ANSERINA XI. THE STATE OF COPPER OF LACCASES I, II AND III

1. Laccases I, II and III were (EC 1.14.18.1) prepared from the mycelium of the ascomycete Podospora anserina. The tetrameric laccase I(mol. wt 340 000, 16 copper atoms) and the monomeric laccases II and II (mol. wt 80 000, 4 copper atoms) have been studied by optical absorption-, circular dichroism-(CD)and electron paramagnetic resonance spectroscopy (EPR). 2. The visible and near ultraviolet difference absorption spectrum, which is apparently identical for all three laccases, shows two maxima at 330 and 610 nm and a shoulder at about 725 nm. The molar extinction coefficients of these bands are 4 times larger for the tetrameric laccase I compared to the monomeric laccases II and III which show values similar to other blue copper-containing oxidases. 3. CD spectra between 300 and 730 nm of the tree laccases are similar and contain at least 5-bands in the oxidized enzyme. If the enzyme is reduced, only a band at 307 nm remains. The molar ellipticity values of these bands are 4 times larger for laccase I than the corresponding bands of laccases II and III. It is inferred that the reducible bands are associated with the Type 1 Cu-2+. 4. In all three laccases the EPR-detectable copper accounts for only about 50% of the total copper content. The 9-GHz and 35-GHz spectra, which are identical for all three laccases, consist of two components of equal intensity. One component shows a rather small copper hyperfine coupling and a small deviation from axial symmetry. It is suggested that this copper is associated with the blue chromophore in analogy to Type 1 Cu-2+ in other blue copper proteins. The other component has a broader hyperfine coupling similar to Type 2 Cu-2+ as found in other copper proteins. The assumption that the experimental spectra result from a superposition of the spectra of equal amounts of Type 1 and Type 2 Cu-2+ has been verified by computer simulation. 5. It is suggested that the copper ions which are not detected by EPR are connected to the absorption band at 330 nm and that these ions are also essential for the function of these laccases.