Franc Pohleven

Submerged cultivation of Ganoderma lucidum biomass and immunostimulatory effects of fungal polysaccharides

Original Ganoderma lucidum strain MZKI G97 isolated from Slovenian forests was cultivated in a liquid substrate based on potato dextrose and olive oil. The influences of inoculum and oxygen partial pressure in batch and fed batch cultivation in a 10-l laboratory stirred tank reactor were studied. Fungal biomass was found to be oxygen and shear sensible. Using a 17% (wet weight) 6 days old vegetative inoculum, 9.6 g l(-1) of dry biomass in batch cultivation and 15.2 g l(-1) in fed batch process were obtained. Extracellular (9.6 g l(-1)) and intracellular (6.3 g l(-1)) polysaccharide fractions were isolated. Extracellular polysaccharide fraction and four intracellular polysaccharide fractions were obtained. Polysaccharides were further separated by ion-exchange, gel and affinity chromatography. The isolated polysaccharides were mainly beta-D-glucanes. Immunostimulatory effects of isolates were tested on induction of cytokine (tumour necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma)) synthesis in primary cultures of human peripheral blood mononuclear cells (PBMC) isolated from a buffy coat. The TNF-alpha inducing activity is comparable with romurtide, which has been used as a supporting therapy in cancer patients treated with radiotherapy and/or chemotherapy.

Effect of the antifungal activity of oxygenated aromatic essential oil compounds on the white-rot Trametes versicolor and the brown-rot Coniophora puteana

Abstract Essential oils and their constituents have a long history of applications as antimicrobial agents, but their use as wood preservatives has not yet been reported. In this study, 22 essential oil phenols, phenol ethers, and aromatic aldehydes have been tested for their antifungal activity against the wood-decaying fungi Trametes versicolor and Coniophora puteana . Minimal inhibitory concentrations of the selected compounds added to potato dextrose agar (PDA) in defined concentrations were determined by a screening test with the agar dilution method. A significant difference in the tolerance of the tested fungi towards the selected compounds was discovered and attributed to the differences in their metabolic characteristics. The influence of the chemical structure of the tested essential oil compounds on their antifungal activity is also discussed.

Pleurotus and Agrocybe hemolysins, new proteins hypothetically involved in fungal fruiting

Novel hemolytic proteins, ostreolysin and aegerolysin, were purified from the fruiting bodies of the edible mushrooms Pleurotus ostreatus and Agrocybe aegerita. Both ostreolysin and aegerolysin have a molecular weight of about 16 kDa, have low isoelectric points of 5.0 and 4.85, are thermolabile, and hemolytic to bovine erythrocytes at nanomolar concentrations. Their activity is impaired by micromolar Hg(2+) but not by membrane lipids and serum low-density lipoproteins (LDL). The sequence of respectively 50 and 10 N-terminal amino acid residues of ostreolysin and aegerolysin has been determined and found to be highly identical with a cDNA-derived amino acid sequence of putative Aa-Pri1 protein from the mushroom A. aegerita, Asp-hemolysin from Aspergillus fumigatus, and two bacterial hemolysin-like proteins expressed during sporulation. We found that ostreolysin is expressed during formation of primordia and fruiting bodies, which is in accord with previous finding that the Aa-Pri1 gene is specifically expressed during fruiting initiation. It is suggestive that the isolated hemolysins play an important role in initial phase of fungal fruiting.

COMPLEXES OF COPPER(II) ACETATE WITH NICOTINAMIDE : PREPARATION, CHARACTERIZATION AND FUNGICIDAL ACTIVITY; CRYSTAL STRUCTURES OF CU2(O2CCH3)4(NIA) AND CU2(O2CCH3)4(NIA)2

Abstract Three new copper (II) acetate complexes with nicotinamide (nia) were synthesized analyzed and characterized by standard chemical and physical methods and tested for fungicidal activity The crystal and molecular structures of the compounds [Cu2 (O2CCH3)4 (nia)] (1B) and [Cu2 (O2CCH3)4 (nia)2] (2) were determined by X-ray diffraction Both consist of binuclear units of bridging tetracarboxylate type however they differ in the bonding mode of nicotinamide molecules They are bonded at the apical positions of the dimers and connect them in an infinite chain in 1B On the other hand the dimers remain isolated in the structure of the compound 2 It seems that compound 1B is the first example where a nicotinamide molecule acts as a bidentate bridging ligand The results of EPR spectra agree with the dimeric nature of the complexes Dissolved in water or DMSO the compounds completely stop mycelial growth at a concentration of 50×10−3 mol l−1 Less concentrated solutions (up to 10×10−3 mol l−1) show weaker fungicidal activity.

Changes in EPR spectra of wood impregnated with copper-based preservatives during exposure to several wood-rotting fungi

Summary The tolerance of various fungi against copper was examined. For this purpose, we impregnated Norway spruce (Picea abies) specimens with two different aqueous solutions: copper(II) octanoate with ethanolamine or copper(II) sulfate (cCu = 1.0 × 10−2 mol/l). Impregnated and unimpregnated test specimens were then exposed to brown rot fungi Antrodia vaillantii and Gloeophyllum trabeum or to white-rot fungi Schizophyllum commune and Trametes versicolor. After 2, 4, 6 and 12 weeks of exposure Electron Paramagnetic Resonance, Atomic Absorption Spectroscopy and mass loss measurements were performed. The results indicate that A. vaillantii, G. trabeum and T. versicolor transform copper(II) sulfate in wood into non-soluble, and therefore non-toxic, copper oxalate. The intensity of this reaction depends on the amount of excreted oxalic acid and was the highest for A. vaillantii and the lowest for T. versicolor. In the presence of ethanolamine, formation of insoluble copper oxalate was not possible and therefore, decay could not proceed. The major portion of copper remained in the wood and only minor amounts were in some cases translocated into nutrient media.

The use of spent brewery grains for Pleurotus ostreatus cultivation and enzyme production.

In the brewing industry, spent brewery grains (SBGs) are byproducts with a low economic value. The potential use of this leftover as a substrate ingredient for Pleurotus ostreatus fruiting body cultivation and enzyme production was evaluated. The best substrate mixture for P. ostreatus mycelium growth comprised 30% wheat bran (WB), 68% beech sawdust (BS) and 2% CaCO3. On the substrates containing SBG, the fastest mycelium growth was observed on the substrate composed of 10% SBG, 20% WB, 68% BS and 2% CaCO3. The highest biological efficiency (51%) of fruiting bodies was determined on the mixtures containing 20% WB, 10% SBG and 2% CaCO3. The SBGs with the addition of WB were also shown to be suitable as a substrate for enzyme production. However, the supplementation levels designate which enzymes are produced and in what amounts.

Degradation of lindane and endosulfan by fungi, fungal and bacterial laccases

The ability of two white-rot fungi (Trametes versicolor and Pleurotus ostreatus) and one brown-rot fungus (Gloeophyllum trabeum) to degrade two organochlorine insecticides, lindane and endosulfan, in liquid cultures was studied and dead fungal biomass was examined for adsorption of both insecticides from liquid medium. Lindane and endosulfan were also treated with fungal laccase and bacterial protein CotA, which has laccase activities. The amount of degraded lindane and endosulfan increased with their exposure period in the liquid cultures of both examined white-rot fungi. Endosulfan was transformed to endosulfan sulphate by T. versicolor and P. ostreatus. A small amount of endosulfan ether was also detected and its origin was examined. Degradation of lindane and endosulfan by a brown rot G. trabeum did not occur. Mycelial biomasses of all examined fungi have been found to adsorb lindane and endosulfan and adsorption onto fungal biomass should therefore be considered as a possible mechanism of pollutant removal when fungal degradation potentials are studied. Bacterial protein CotA performed more efficient degradation of lindane and endosulfan than fungal laccase and has shown potential for bioremediation of organic pollutants.

Complexes of copper(II) carboxylates with 2-aminoethanol - syntheses, characterization and fungicidal activity; crystal structure of Cu(O2CC8H17)2(NH2C2H4OH)2

Abstract Complexes of copper(II) octanoate, nonanoate and decanoate with 2-aminoethanol (ethanolamine) have been synthesized and characterized. The crystal structure of Cu(O2CC8H17)2(NH2C2H4OH)2 consists of mononuclear units with pairs of amino nitrogen atoms and deprotonated nonanoato oxygen atoms forming a square coordination plane around the central copper. The hydroxyl oxygen atoms from the 2-aminoethanol ligands are positioned axially with respect to the coordination plane. Results of screening for fungicidal activity against the wood-rotting fungus Trametes versicolor (L. ex Fr.) Pilat show that the complexes totally inhibit fungal growth at a concentration of 1.0 × 10−3 mol/1 but only partially at a concentration of 5.0 × 10−4 mol/1.

Influence of carboxylic acids on fixation of copper in wood impregnated with copper amine based preservatives

The importance of copper/amine based preservatives is increasing. Leaching of copper from wood preserved with these solutions is still higher than leaching from wood impregnated with copper chromium ones. In order to decrease leaching, different carboxylic acids (octanoic, 2-ethylhexanoic, decanoic) were added to copper/amine/boron aqueous solutions. An experiment of leaching of copper from Norway spruce was performed according to the modified standard procedure (EN 1250). Results confirmed that carboxylic acids significantly improve copper fixation. The best fixation was determined in specimens impregnated with the preservative solutions consisting of copper, ethanolamine, boric acid and octanoic acid. From such wood, only 1.6% of copper was leached.

Performance of Waterborne Cu(II) Octanoate/Ethanolamine Wood Preservatives

Summary Various aqueous wood preservative solutions containing Cu(II) in the form of copper(II) sulphate or copper(II) octanoate, ethanolamine and in one case octanoic acid were investigated by spectrophotometry, polarography and Electron Paramagnetic Resonance (EPR) spectroscopy. Results have shown the same coordination environment around Cu(II) in all solutions with ethanolamine. Computer simulation of the EPR spectra also revealed that the coordination in the first coordination sphere of copper is the same at low concentration of ethanolamine. At 20% ethanolamine concentration, a mixture of two complexes (one with two nitrogens and the other with three) could be detected. The active compound in the investigated ethanolamine containing solutions is the same when previously synthesised copper(II) octanoate was used, or when copper(II) sulphate and octanoic acid were utilized instead. Fungicidal and leaching experiments with the treated wood resulted in the same conclusion: it is not necessary to use pre-synthesised copper(II) octanoate for the preparation of waterborne copper/ethanolamine wood preservatives. Preservative preparation time and costs can be reduced by simply dissolving copper(II) sulphate and octanoic acid in aqueous ethanolamine solutions.