Penicillium subrubescens, a new species efficiently producing inulinase.

Inulin is a reserve carbohydrate in about 15 % of the flowering plants and is accumulated in underground tubers of e.g. chicory, dahlia and Jerusalem artichoke. This carbohydrate consists of linear chains of ß-(2,1)-linked fructose attached to a sucrose molecule. Inulinases hydrolyse inulin into fructose and glucose. To find efficient inulin degrading fungi, 126 fungal strains from the Fungal Biotechnology Culture Collection (FBCC) at University of Helsinki and 74 freshly isolated strains from soil around Jerusalem artichoke tubers were screened in liquid cultures with inulin as a sole source of carbon or ground Jerusalem artichoke tubers, which contains up to 19 % (fresh weight) inulin. Inulinase and invertase activities were assayed by the dinitrosalicylic acid (DNS) method and a freshly isolated Penicillium strain originating from agricultural soil (FBCC 1632) was the most efficient inulinase producer. When it was cultivated at pH 6 and 28 °C in 2 litre bioreactors using inulin and Jerusalem artichoke as a carbon source, inulinase and invertase activities were on day 4 7.7 and 3.1 U mL(-1), respectively. The released sugars analysed by TLC and HPLC showed that considerable amounts of fructose were released while the levels of oligofructans were low, indicating an exoinulinase type of activity. Taxonomic study of the inulinase producing strain showed that this isolate represents a new species belonging in Penicillium section Lanata-divaricata. This new species produces a unique combination of extrolites and is phenotypically and phylogenetically closely related to Penicillium pulvillorum. We propose the name Penicillium subrubescens sp. nov. (CBS 132785(T) = FBCC 1632(T)) for this new species.

Heavy metals in bottom sediments of Lake Umbozero in Murmansk Region, Russia.

Sediment cores collected from different locations of Lake Umbozero were studied with respect to concentration and mobility of trace and heavy metals Co, Cu, Fe, Mn, Ni, Pb, U, and Zn. Lake Umbozero is the second largest lake in the Murmansk Region and subjected to contamination by air-borne emissions and river transportation from the nearby metallurgical and mining industries. Unlike its neighboring, more industry-prone Lake Imandra, Lake Umbozero is relatively unexplored with respect to its state of pollution. In our study, metal distribution in sediments was found to vary with respect to the cores, although in general the concentrations were at the same level throughout the lake indicating uniform horizontal distribution of metals. When compared to Lake Imandra, the concentrations of most of the metals studied were significantly lower and represented the levels in sediments measured in lakes of Kola Peninsula located further off from industrial pollutant sources. An exception was Pb the concentration of which was at the same level as in Lake Imandra, probably due to long-distance transport. Sediment layers were subjected to four-step sequential extraction procedure to reveal the metal distribution in soluble, exchangeable, acid-soluble, and residual fractions. Indicative of their potential higher lability, Mn, U, and Zn were generally found in exchangeable fraction; as also Mn and U extensively in the acid-soluble fraction.

Mineralization of 14C-labelled synthetic lignin and extracellular enzyme activities of the wood-colonizing ascomycetes Xylaria hypoxylon and Xylaria polymorpha.

Two wood-dwelling ascomycetes, Xylaria hypoxylon and Xylaria polymorpha, were isolated from rotting beech wood. Lignin degradation was studied following the mineralization of a synthetic [formula: see text]-labelled lignin in solid and liquid media. Approximately 9% of the synthetic lignin was mineralized by X. polymorpha during the growth on beech wood meal, and the major fraction (65.5%) was polymerized into water- and dioxan-insoluble material. Both fungi produced laccase (up to 1,200 U l-1) in an agitated complex medium based on tomato juice; peroxidase activity (<80 U l-1) was only detected for X. polymorpha in soybean meal suspension. The enzymatic attack of X. polymorpha on beech wood resulted in the formation of three fractions of water-soluble lignocellulose fragments with molecular masses of 200, 30 (major fraction) and 3 kDa, as demonstrated by high-performance size exclusion chromatography. This fragment pattern differs considerably from that of the white-rot fungus Bjerkandera adusta, which preferentially released smaller lignocellulose fragments (0.8 kDa). The finding that X. polymorpha produced large lignocellulose fragments, along with the fact that high levels of hydrolytic enzymes (esterase 630 U l-1, xylanase 120 U l-1) were detected, indicates the cleavage of bonds between the lignin and hemicellulose moieties.

A rapid method to quantify pro-oxidant activity in cultures of wood-decaying white-rot fungi.

A new, rapid method for evaluation of lipid peroxidation promoting (pro-oxidant) activity in cultures of wood-decaying fungi was developed. The method is based on measurement of the rate of oxygen consumption in the reaction of linoleic acid peroxidation initiated by fungal culture filtrates. The liquid cultures of the white-rot fungi Bjerkandera adusta and Phanerochaete chrysosporium grown on wheat straw-containing glucose-peptone-corn steep liquor medium possessed significant levels of the pro-oxidant activity. Other white-rot fungi producing manganese peroxidase (MnP) were also found to show the activity. MnP demonstrated a crucial role as the major pro-oxidant agent in the fungal cultures. The total pro-oxidant activity may be considered as net result of the peroxidation by MnP and the inhibition by antioxidant compounds present in the fungal culture fluids.

Lignin degradation in a compost environment by the deuteromycete Paecilomyces inflatus.

Two strains of the deuteromycete Paecilomyces inflatus were isolated from compost samples consisting of municipal wastes, paper and wood chips. Lignin degradation by P. inflatus was studied following the mineralization of a synthetic (14)C(beta)-labeled lignin (side-chain labeled dehydrogenation polymer, DHP). Approximately 6.5% of the synthetic lignin was mineralized during solid-state cultivation of the fungus in autoclaved compost; and 15.5% was converted into water-soluble fragments. Laccase was the only ligninolytic enzyme detectable when the isolates were grown in autoclaved compost. Production of the enzyme was growth-associated and dependent on the culture conditions. The optimal pH for laccase production was between 4.5 and 5.5 and the optimal temperature was around 30 degrees C. Activity levels of laccase increased in the presence of low-molecular-mass aromatic compounds, such as veratryl alcohol, veratric acid, vanillin and vanillic acid.

Removal and mineralization of polycyclic aromatic hydrocarbons by litter-decomposing basidiomycetous fungi.

Nine strains of litter-decomposing fungi, representing eight species of agaric basidiomycetes, were tested for their ability to remove a mixture of three polycyclic aromatic hydrocarbons (PAHs) (total 60 mg l(-1)) comprising anthracene, pyrene and benzo(a)pyrene (BaP) in liquid culture. All strains were able to convert this mixture to some extent, but considerable differences in degradative activity were observed depending on the species, the Mn(II) concentration, and the particular PAH. Stropharia rugosoannulata was the most efficient degrader, removing or transforming BaP almost completely and about 95% of anthracene and 85% of pyrene, in cultures supplemented with 200 micro M Mn(II), within 6 weeks. In contrast less than 40, 18, and 50% BaP, anthracene and pyrene, respectively, were degraded in the absence of supplemental Mn(II). In the case of Stropharia coronilla, the presence of Mn(II) led to a 20-fold increase of anthracene conversion. The effect of manganese could be attributed to the stimulation of manganese peroxidase (MnP). The maximum activity of MnP increased in S. rugosoannulata cultures from 10 U l(-1) in the absence of Mn(II) to 320 U l(-1) in Mn(II)-supplemented cultures. The latter degraded about 6% of a (14)C-labeled BaP into (14)CO(2) whereas only 0.7% was mineralized in the absence of Mn(II). In solid-state straw cultures, S. rugosoannulata, S. coronilla and Agrocybe praecox mineralized between 4 and 6% of (14)C-labeled BaP within 12 weeks.

Conversion of milled pine wood by manganese peroxidase from Phlebia radiata.

Purified manganese peroxidase (MnP) from the white-rot basidiomycete Phlebia radiata was found to convert in vitro milled pine wood (MPW) suspended in an aqueous reaction solution containing Tween 20, Mn(2+), Mn-chelating organic acid (malonate), and a hydrogen peroxide-generating system (glucose-glucose oxidase). The enzymatic attack resulted in the polymerization of lower-molecular-mass, soluble wood components and in the partial depolymerization of the insoluble bulk of pine wood, as demonstrated by high-performance size exclusion chromatography (HPSEC). The surfactant Tween 80 containing unsaturated fatty acid residues promoted the disintegration of bulk MPW. HPSEC showed that the depolymerization yielded preferentially lignocellulose fragments with a predominant molecular mass of ca. 0.5 kDa. MnP from P. radiata (MnP3) turned out to be a stable enzyme remaining active for 2 days even at 37 degrees C with vigorous stirring, and 65 and 35% of the activity applied was retained in Tween 20 and Tween 80 reaction mixtures, respectively. In the course of reactions, major part of the Mn-chelator malonate was decomposed (85 to 87%), resulting in an increase of pH from 4.4 to >6.5. An aromatic nonphenolic lignin structure (beta-O-4 dimer), which is normally not attacked by MnP, was oxidizible in the presence of pine wood meal. This finding indicates that certain wood components may promote the degradative activities of MnP in a way similar to that promoted by Tween 80, unsaturated fatty acids, or thiols.

Biodegradation of radiolabelled synthetic lignin (14C-DHP) and mechanical pulp in a compost environment.

Mineralization of radioactive synthetic lignin (14C-DHP) was studied in a compost environment at 35, 50 and 58 degrees C. Compost samples were successively extracted with water, dioxane and alkali, and the molecular weight distribution of some extracts was determined by gel permeation chromatography (GPC). Biodegradation of lignin-containing spruce groundwood (SGW) and pine sawdust was concurrently determined in controlled composting tests by measuring evolved CO2. The temperatures were the same as in the 14C-DHP mineralization experiment and bleached kraft paper, with a lignin content of 0.2%, was used as a reference. The mineralization of 14C-DHP was relatively high (23-24%) at 35 degrees C and 50 degrees C, although the mixed population of compost obviously lacks the most effective lignin degraders. At 58 degrees C the mineralization of 14C-DHP, as well as the biodegradation of SGW and sawdust, was very low, indicating that the lignin-degrading organisms of compost were inactivated at this temperature. SGW was poorly biodegradable (<40%) in controlled composting tests compared with kraft paper (77-86%) at all temperatures, which means that lignin inhibits the degradation of carbohydrates. During the incubation, water-soluble degradation products, mainly monomers and dimers, and the original 14C-DHP were either mineralized or bound to humic substances. A substantial fraction of 14C-DHP was incorporated into humin or other insolubles.

Characteristics and N-terminal amino acid sequence of manganese peroxidase from solid substrate cultures of Agaricus bisporus.

Extracellular manganese peroxidase (MnP) was purified from the compost extract of Agaricus bisporus using anion exchange chromatography, gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Two forms (MnP1 and MnP2) were separated by isoelectric focusing and their isoelectric points were determined to be 3.25 (MnP1) and 3.3 (MnP2). Both forms had a molecular mass of 40 kDa. The first 25 amino acids of the N-terminal end of MnP1 sequence was found to share 68% identity with a Pleurotus ostreatus and a P. eryngii MnP. Lignin peroxidase was not detected during any of the steps in the purification process. In liquid cultures with both soluble and insoluble carbon sources in defined medium (D-glucose, glycerol, Whatman CC-41 microcrystalline cellulose or Solka-floc cellulose) MnP protein was detected in culture fluid by Western blot, but no MnP activity could be detected. A. bisporus appears to be in the group of ligninolytic fungi which do not produce lignin peroxidase.

Silver stained polyacrylamide gels and fluorescence-based automated capillary electrophoresis for detection of amplified fragment length polymorphism patterns obtained from white-rot fungi in the genus Trametes.

Silver stained denaturing polyacrylamide gels (PAGEs) and fluorescent denaturing automated capillary electrophoresis (CE) were used to detect amplified fragment length polymorphism (AFLP) patterns obtained from white-rot fungi belonging to the genus Trametes. AFLP fingerprinting detected by the fluorescence-based method as well as by silver staining showed a high discriminatory power in differentiating nine strains of Trametes ochracea, nine strains of Trametes hirsuta and ten isolates of Trametes versicolor. UPGMA dendrograms derived from fluorescently labelled and silver stained AFLP patterns were similar, but a few differences were detected especially in the clustering of T. ochracea and T. hirsuta strains. Compared to silver-stained AFLP, detection of fluorescent AFLP was fast, reliable and easy to perform and it facilitated surveying with a computerized analysis system. Fluorescent CE seems to be well suited for studying similarity between Trametes species.