Biochemical and molecular characterization of an atypical manganese peroxidase of the litter-decomposing fungus Agrocybe praecox.

Agrocybe praecox is a litter-decomposing Basidiomycota species of the order Agaricales, and is frequently found in forests and open woodlands. A. praecox grows in leaf-litter and the upper soil and is able to colonize bark mulch and wood chips. It produces extracellular manganese peroxidase (MnP) activities and mineralizes synthetic lignin. In this study, the A. praecox MnP1 isozyme was purified, cloned and enzymatically characterized. The enzyme catalysed the oxidation of Mn(2+) to Mn(3+), which is the specific reaction for manganese-dependent class II heme-peroxidases, in the presence of malonate as chelator with an activity maximum at pH 4.5; detectable activity was observed even at pH 7.0. The coding sequence of the mnp1 gene demonstrates a short-type of MnP protein with a slightly modified Mn(2+) binding site. Thus, A. praecox MnP1 may represent a novel group of atypical short-MnP enzymes. In lignocellulose-containing cultures composed of cereal bran or forest litter, transcription of mnp1 gene was followed by quantitative real-time RT-PCR. On spruce needle litter, mnp1 expression was more abundant than on leaf litter after three weeks cultivation. However, the expression was constitutive in wheat and rye bran cultures. Our data show that the atypical MnP of A. praecox is able to catalyse Mn(2+) oxidation, which suggests its involvement in lignocellulose decay by this litter-decomposer.

Bioluminescent yeast assay for detection of organotin compounds.

Organotin compounds are toxic and endocrine disrupting compounds, which have been intensively used as antifouling paints for ship hulls and thus are widely spread in the environment. They are suspected to cause imposex, the formation of male characteristics in female gastropods, because of the activation of retinoid X receptor (RXR) at very low environmental concentrations. Here we report the development and optimization of a bioluminescent yeast assay for the detection of organotin compounds based on the interaction with a hybrid RXR and subsequent expression of a reporter luciferase gene. This assay is highly specific toward organotin compounds and natural ligands of the RXR. It detects tributyltin and triphenyltin in nanomolar concentrations (detection limits were found to be 30 nM and 110 nM, respectively) and allows small-scale high-throughput analyses. Furthermore it was possible to measure tributyltin directly in untreated spiked sediments. Thus, the results provided within one working day can be used for the assessment of bioavailability and mixture effect of organotin compounds in environmental samples.

Substrate oxidation by dye-decolorizing peroxidases (DyPs) from wood- and litter-degrading agaricomycetes compared to other fungal and plant heme-peroxidases.

Catalytic and physicochemical properties of representative fungal dye-decolorizing peroxidases (DyPs) of wood- (WRF) and litter-decomposing white-rot fungi (LDF) are summarized and compared, including one recombinant Mycetinis scorodonius DyP (rMscDyP; LDF), the wild-type Auricularia auricula-judae DyP (AauDyP; WRF), and two new DyPs secreted by the jelly fungi Exidia glandulosa (EglDyP; WRF) and Mycena epipterygia (MepDyP; LDF). Homogeneous preparations of these DyPs were obtained after different steps of fast protein liquid chromatography, and they increase the total number of characterized fungal DyP proteins to eight. The peptide sequences of AauDyP, MepDyP, and EglDyP showed highest homologies (52-56%) to the DyPs of M. scorodonius. Five out of the eight characterized fungal DyPs were used to evaluate their catalytic properties compared to classic fungal and plant heme peroxidases, namely lignin peroxidase of Phanerochaete chrysosporium (PchLiP; WRF), versatile peroxidase of Bjerkandera adusta (BadVP; WRF), and generic peroxidases of Coprinopsis cinerea (CiP) and Glycine max (soybean peroxidase=SBP). All DyPs tested possess unique properties regarding the stability at low pH values: 50-90% enzymatic activity remained after 4-h exposition at pH 2.5, and the oxidation of nonphenolic aromatic substrates (lignin model compounds) was optimal below pH 3. Furthermore, all DyPs efficiently oxidized recalcitrant dyes (e.g., Azure B) as well as the phenolic substrate 2,6-dimethoxyphenol. Thus, DyPs combine features of different peroxidases on the functional level and may be part of the biocatalytic system secreted by fungi for the oxidation of lignin and/or toxic aromatic compounds.

Evaluation of bone growth around bioactive glass S53P4 by scanning acoustic microscopy co-registered with optical interferometry and elemental analysis.

Bioactive glass (BAG) is a bone substitute that can be used in orthopaedic surgery. Following implantation, the BAG is expected to be replaced by bone via bone growth and gradual degradation of the BAG. However, the hydroxyapatite mineral forming on BAG resembles bone mineral, not providing sufficient contrast to distinguish the two in X-ray images. In this study, we co-registered coded-excitation scanning acoustic microscopy (CESAM), scanning white light interferometry (SWLI), and scanning electron microscopy with elemental analysis (Energy Dispersive X-ray Spectroscopy) (SEM-EDX) to investigate the bone growth and BAG reactions on a micron scale in a rabbit bone ex vivo. The acoustic impedance map recorded by the CESAM provides high elasticity-associated contrast to study materials and their combinations, while simultaneously producing a topography map of the sample. The acoustic impedance map correlated with the elemental analysis from SEM-EDX. SWLI also produces a topography map, but with higher resolution than CESAM. The two topography maps (CESAM and SWLI) were in good agreement. Furthermore, using information from both maps simultaneously produced by the CESAM (acoustic impedance and topography) allowed determining regions-of-interest related to bone formation around the BAG with greater ease than from either map alone. CESAM is therefore a promising tool for evaluating the degradation of bone substitutes and the bone healing process ex vivo.

Meat and bone meal as a novel biostimulation agent in hydrocarbon contaminated soils.

Soil contamination with diesel oil is frequent and methods to improve remediation of diesel oil contaminated soils are urgently needed. The aim of the current study was to assess the potential of meat and bone meal (MBM) as a biostimulation agent to enhance diesel oil degradation in contaminated soils collected from southern Finland. MBM (2% w/w) increased oil degradation in soils when compared to natural attenuation. The increase was comparable to soils treated with a traditional fertilizer (urea). Soil pH increased rapidly in urea treated soil but remained at the level of natural attenuation in MBM treated soil, suggesting that in large-scale experiments MBM treated soils avoid the usual negative impact of urea on soil pH and ultimately microbial degradation. These results indicate that MBM addition enhances diesel oil degradation, and that MBM speeds up ex situ bioremediation of oil contaminated soils.

Morphological and molecular identification of three new species of Tomentella from Finland.

Three new species of Tomentella (Thelephorales) from Finland, T. globosa, T. lammiensis, and T. longisterigmata, are described and illustrated with morphological characteristics and nuc rDNA ITS1-5.8S-ITS2 sequences. T. globosa is characterized by mucedinoid, pale to dark brown basidiocarps adherent to the substrate, generative hyphae with clamps and rarely with simple septa, and echinulate, globose basidiospores (echinuli up to 1.5 µm long). T. lammiensis is characterized by mucedinoid, oxide yellow to golden brown basidiocarps adherent to the substrate, generative hyphae with clamps and rarely with simple septa, and echinulate, ellipsoid, triangular, or lobbed basidiospores (echinuli up to 2 µm long). T. longisterigmata is characterized by mucedinoid, dark brown to chestnut basidiocarps separable from the substrate, generative hyphae clamped and rarely with simple septa, the long basidial sterigmata (7-11 µm long), and echinulate, globose basidiospores (echinuli up to 2 µm long). An absence of rhizomorphs and cystidia is their common morphological feature. Molecular analyses by maximum likelihood, maximum parsimony, and Bayesian analyses confirm the phylogenetic position of these three new species. The discriminating characters of these new species and their closely related species are discussed in this study, and a key to the species from Finland is provided.

Differential degradation of oak (Quercus petraea) leaf litter by litter-decomposing basidiomycetes.

Due to production of lignocellulose-degrading enzymes, saprotrophic litter-decomposing basidiomycetes can significantly contribute to the turnover of soil organic matter. The production of lignin and polysaccharide-degrading enzymes and changes in the chemical composition of litter was studied with Marasmius quercophilus, Mycena inclinata and Pholiota lenta, three basidiomycete species typical of oak (Quercus petraea) forests. Within 12weeks of incubation, M. inclinata decomposed 33%, M. quercophilus 36% and P. lenta 48% of the substrate dry mass. All fungi produced laccase and Mn-peroxidase and none of them produced lignin peroxidase or Mn-independent peroxidases. M. inclinata and M. quercophilus produced considerable laccase activity, while production by P. lenta was low. M. quercophilus and P. lenta produced most Mn-peroxidase at the beginning of the experiment, while the production by M. inclinata was more stable in time. Endo-1,4-beta-xylanase exhibited the highest activity among endocleaving glycosyl hydrolases while 1,4-beta-glucosidase was the main exocleaving enzyme. All fungi decreased the C:N ratio of the litter from 27 to 13-17 and M. inclinata and M. quercophilus also decreased the lignin content. Analytical pyrolysis of decayed litter showed changes in litter composition similar to those caused by white-rot fungi during wood decay, e.g. a decrease in the syringyl/guaiacyl lignin ratio. These changes were more pronounced in M. inclinata and M. quercophilus. The results indicate that different litter-decomposing fungi can cause substantial litter transformation despite considerable differences in the production of lignocellulose-degrading enzymes.

Influence of Pb contamination in boreal forest soil on the growth and ligninolytic activity of litter-decomposing fungi.

Lignin mineralization activity of three basidiomycetous litter-decomposing fungi (LDF) was studied with humus layer samples taken from a boreal forest soil. The total Pb concentration in the samples was 32,000 mg kg(-1) and water soluble Pb 67 mg kg(-1). Synthetic lignin mineralization by Collybia dryophila and Clitocybe (Lepista) nebularis was strongly inhibited, whereas Stropharia coronilla was more tolerant to Pb stress in soil and liquid cultures. Purified laccases maintained their activity and purified MnPs remained partly active up to a concentration of 1450 mg Pb l(-1). High concentrations of Pb inhibited the growth of LDF and affected the activity of ligninolytic enzymes, but the extent of inhibition varied among different LDF species. In consequence, Pb contamination in soil may have a negative impact on recycling of organic carbon.

Potential of cometabolic transformation of polysaccharides and lignin in lignocellulose by soil Actinobacteria.

While it is known that several Actinobacteria produce enzymes that decompose polysaccharides or phenolic compounds in dead plant biomass, the occurrence of these traits in the environment remains largely unclear. The aim of this work was to screen isolated actinobacterial strains to explore their ability to produce extracellular enzymes that participate in the degradation of polysaccharides and their ability to cometabolically transform phenolic compounds of various complexities. Actinobacterial strains were isolated from meadow and forest soils and screened for their ability to grow on lignocellulose. The potential to transform (14)C-labelled phenolic substrates (dehydrogenation polymer (DHP), lignin and catechol) and to produce a range of extracellular, hydrolytic enzymes was investigated in three strains of Streptomyces spp. that possessed high lignocellulose degrading activity. Isolated strains showed high variation in their ability to produce cellulose- and hemicellulose-degrading enzymes and were able to mineralise up to 1.1% and to solubilise up to 4% of poplar lignin and to mineralise up to 11.4% and to solubilise up to 64% of catechol, while only minimal mineralisation of DHP was observed. The results confirm the potential importance of Actinobacteria in lignocellulose degradation, although it is likely that the decomposition of biopolymers is limited to strains that represent only a minor portion of the entire community, while the range of simple, carbon-containing compounds that serve as sources for actinobacterial growth is relatively wide.

Fungal enzyme production and biodegradation of polychlorinated dibenzo-p-dioxins and dibenzofurans in contaminated sawmill soil.

The current treatment method for PCDD/F-contaminated soil, which fulfils the requirements for POP soils, is incineration at high temperature. In this study, we investigated if bioaugmentation with fungal inoculum or treatment with manganese peroxidase (MnP) enzyme preparation could be used instead. The main source of PCDD/F contamination in Finland has been the national production and use of a chlorophenol containing wood preservative, which contained PCDD/Fs as impurities. Therefore, historically contaminated soils from three sawmill sites were used in the experiments. In bioaugmentation experiments with living fungal mycelia, enzyme production, CO2 production and degradation of chlorinated dioxins were measured. When cell free MnP preparation was added to the soil, it was likewise important to follow how enzyme activity was maintained in the soil. As a result of this study, we showed that fungi were able to efficiently degrade PCDD/F, but surprisingly the addition of MnP preparation did not have any effect to the PCDD/F concentration. However, substantial amounts of MnP activity were found in the soil still after 10d of incubation. Treatment with either Stropharia rugosoannulata or Phanerochaete velutina resulted in 62-64% decrease in WHO-TEQ value in 3months. One critical factor for efficient biodegradation was strong growth of fungal mycelia in non-sterile contaminated soil.

Mycoremediation of wood and soil from an old sawmill area contaminated for decades.

We investigated the potential of white-rot and litter-decomposing fungi for the treatment of soil and wood from a sawmill area contaminated with aged chlorinated phenols, dibenzo-p-dioxins and furans (PCDD/F). Eight screening assays with emphasis on application of non-sterile conditions were carried out in order to select the strains with capability to withstand indigenous microbes and contamination. Nine fungi were then selected for degrading pentachlorophenol (PCP), and 2,3,4,6-tetrachlorophenol (2,3,4,6-TeCP) and mineralizing radiolabelled pentachlorophenol ((14)C-PCP) in non-sterile soil or wood during 15 weeks of incubation. Soil indigenous microbes and fungal inoculated soil (fungal inoculum+indigenous microbes) achieved similar degradation of PCP and 2,3,4,6-TeCP and mineralization of (14)C-PCP. However, the mineralization rate of (14)C-PCP by indigenous microbes was much slower than that boosted by fungal inoculum. The litter-decomposing fungus (LDF) Stropharia rugosoannulata proved to be a suitable fungus for soil treatment. This fungus mineralized 26% of (14)C-PCP and degraded 43% of 2,3,4,6-TeCP and 73% of PCP. Furthermore, S. rugosoannulata attained 13% degradation of PCDD/F (expressed as WHO-Toxic Equivalent). In wood, white-rot fungi grew and degraded chlorophenols better than LDF. No efficient indigenous degraders were present in wood. Interestingly, production of toxic chlorinated organic metabolites (anisoles and veratroles) by LDF in wood was negligible.

Fate of bisphenol A during treatment with the litter-decomposing fungi Stropharia rugosoannulata and Stropharia coronilla.

Bisphenol A is an endocrine disrupting compound, which is ubiquitous in the environment due to its wide use in plastic and resin production. Seven day old cultures of the litter-decomposing fungus Stropharia coronilla removed the estrogenic activity of bisphenol A (BPA) rapidly and enduringly. Treatment of BPA with purified neutral manganese peroxidase (MnP) from this fungus also resulted in 100% reduction of estrogenic activity, as analyzed using a bioluminescent yeast assay, and in the formation of polymeric compounds. In cultures of Stropharia rugosoannulata, estrogenic activity also quickly disappeared but temporarily re-emerged in the further course of cultivation. LC-MS analysis of the extracted estrogenic culture liquid revealed [M-H](-) ions with m/z values of 219 and 235. We hypothesize that these compounds are ring fission products of BPA, which still exhibit one intact hydroxyphenyl group to interact with estrogen receptors displayed by the yeast.

Scots pine (Pinus sylvestris) bark composition and degradation by fungi: potential substrate for bioremediation.

The composition of Scots pine bark, its degradation, and the production of hydrolytic and ligninolytic enzymes were evaluated during 90 days of incubation with Phanerochaete velutina and Stropharia rugosoannulata. The aim was to evaluate if pine bark can be a suitable fungal substrate for bioremediation applications. The original pine bark contained 45% lignin, 25% cellulose, and 15% hemicellulose. Resin acids were the most predominant lipophilic extractives, followed by sitosterol and unsaturated fatty acids, such as linoleic and oleic acids. Both fungi degraded all main components of bark, specially cellulose (79% loss by P. velutina). During cultivation on pine bark, fungi also degraded sitosterol, produced malic acid, and oxidated unsaturated fatty acids. The most predominant enzymes produced by both fungi were cellulase and manganese peroxidase. The results indicate that Scots pine bark supports enzyme production and provides nutrients to fungi, thus pine bark may be suitable fungal substrate for bioremediation.

Enhancement of bioconversion of high-molecular mass polycyclic aromatic hydrocarbons in contaminated non-sterile soil by litter-decomposing fungi.

With the focus on alternative microbes for soil-bioremediation, 18 species of litter-decomposing basidiomycetous fungi were screened for their ability to grow on different lignocellulosic substrates including straw, flax and pine bark as well as to produce ligninolytic enzymes, namely laccase and manganese peroxidase. Following characteristics have been chosen as criteria for the strain selection: (i) the ability to grow at least on one of the mentioned materials, (ii) production of either of the ligninolytic enzymes and (iii) the ability to invade non-sterile soil. As the result, eight species were selected for a bioremediation experiment with an artificially contaminated soil (total polycyclic aromatic hydrocarbon (PAH) concentration 250 mg/kg soil). Up to 70%, 86% and 84% of benzo(a)anthracene, benzo(a)pyrene, and dibenzo(a,h)anthracene, respectively, were removed in presence of fungi while the indigenous microorganisms converted merely up to 29%, 26% and 43% of these compounds in 30 days. Low molecular-mass PAHs studied were easily degraded by soil microbes and only anthracene degradation was enhanced by the fungi as well. The agaric basidiomycetes Stropharia rugosoannulata and Stropharia coronilla were the most efficient PAH degraders among the litter-decomposing species used.

Modification of humic acids by the compost-dwelling deuteromycete Paecilomyces inflatus.

The soil mold Paecilomyces inflatus is capable of modifying and partially mineralizing synthetic and natural humic acids (HAs) in compost environments. HA degradation studies using a synthetic HA (14C-HA) in autoclaved compost microcosms showed that, after 12 weeks of cultivation, P. inflatus mineralized approximately 5% of the 14C-labeled HA to 14CO2, while 6% of the 14C-HA was converted into 14C-labeled water-soluble fragments (fulvic-acid-like fraction). About 40% was still present as NaOH-soluble HA representing unmodified or only slightly modified humic material (compared with 60% in the controls). Modification of natural HAs extracted from compost was followed by their partial decolorization (30%) in liquid cultures of P. inflatus. Bleaching of the medium was accompanied by moderate changes in the molecular mass distribution of both the HA and fulvic-acid fractions, which were analyzed with high-performance size exclusion chromatography. HA modification was most pronounced during the primary growth phase of the fungus and was associated with increased laccase activity.

Involvement of lipid peroxidation in the degradation of a non-phenolic lignin model compound by manganese peroxidase of the litter-decomposing fungus Stropharia coronilla.

Culture liquids of the litter-decomposing basidiomycete Stropharia coronilla showed pro-oxidant activity promoting the peroxidation of linoleic acid. This activity depended on the presence of manganese peroxidase (MnP) in the fungal culture. Pro-oxidant activity maxima coincided with maximum MnP activities during the separation of extracellular proteins by anion-exchange chromatography. Purified MnP1 showed substantial pro-oxidant activity in the presence of acetate and Mn2+ ions, even without the addition of hydrogen peroxide. A non-phenolic beta-O-4 lignin model compound [LMC; 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-1,3-dihydroxypropane] was partially oxidized in an in vitro reaction system developing MnP-dependent lipid peroxidation. The chelating organic acids malonate and tartrate noticeably inhibited both the peroxidation of linoleic acid and the conversion of LMC in the system. The major product of the LMC oxidation was 1-(3,4-dimethoxyphenyl)-1-oxo-2-(2-methoxyphenoxy)-3-hydroxypropane; in addition, small amounts of 3,4-dimethoxybenzaldehyde (veratraldehyde) and 3,4-dimethoxybenzoic (veratric) acid were detected. Thus, MnP-initiated lipid peroxidation may be involved in the degradation of recalcitrant non-phenolic lignin substructures by litter-decomposing fungi similar to MnPs of wood-decaying fungi.

Degradation of benzo[a]pyrene by the litter-decomposing basidiomycete Stropharia coronilla: role of manganese peroxidase.

The litter-decomposing basidiomycete Stropharia coronilla, which preferably colonizes grasslands, was found to be capable of metabolizing and mineralizing benzo[a]pyrene (BaP) in liquid culture. Manganese(II) ions (Mn(2+)) supplied at a concentration of 200 micro M stimulated considerably both the conversion and the mineralization of BaP; the fungus metabolized and mineralized about four and twelve times, respectively, more of the BaP in the presence of supplemental Mn(2+) than in the basal medium. This stimulating effect could be attributed to the ligninolytic enzyme manganese peroxidase (MnP), whose activity increased after the addition of Mn(2+). Crude and purified MnP from S. coronilla oxidized BaP efficiently in a cell-free reaction mixture (in vitro), a process which was enhanced by the surfactant Tween 80. Thus, 100 mg of BaP liter(-1) was converted in an in vitro reaction solution containing 1 U of MnP ml(-1) within 24 h. A clear indication was found that BaP-1,6-quinone was formed as a transient metabolite, which disappeared over the further course of the reaction. The treatment of a mixture of 16 different polycyclic aromatic hydrocarbons (PAHs) selected by the U.S. Environmental Protection Agency as model standards for PAH analysis (total concentration, 320 mg liter(-1)) with MnP resulted in concentration decreases of 10 to 100% for the individual compounds, and again the stimulating effect of Tween 80 was observed. Probably due to their lower ionization potentials, poorly bioavailable, high-molecular-mass PAHs such as BaP, benzo(g,h,i)perylene, and indeno(1,2,3-c,d)pyrene were converted to larger extents than low-molecular-mass ones (e.g., phenanthrene and fluoranthene).

Degradation of humic acids by the litter-decomposing basidiomycete Collybia dryophila.

The basidiomycete Collybia dryophila K209, which colonizes forest soil, was found to decompose a natural humic acid isolated from pine-forest litter (LHA) and a synthetic (14)C-labeled humic acid ((14)C-HA) prepared from [U-(14)C]catechol in liquid culture. Degradation resulted in the formation of polar, lower-molecular-mass fulvic acid (FA) and carbon dioxide. HA decomposition was considerably enhanced in the presence of Mn(2+) (200 microM), leading to 75% conversion of LHA and 50% mineralization of (14)C-HA (compared to 60% and 20%, respectively, in the absence of Mn(2+)). There was a strong indication that manganese peroxidase (MnP), the production of which was noticeably increased in Mn(2+)-supplemented cultures, was responsible for this effect. The enzyme was produced as a single protein with a pI of 4.7 and a molecular mass of 44 kDa. During solid-state cultivation, C. dryophila released substantial amounts of water-soluble FA (predominantly of 0.9 kDa molecular mass) from insoluble litter material. The results indicate that basidiomycetes such as C. dryophila which colonize forest litter and soil are involved in humus turnover by their recycling of high-molecular-mass humic substances. Extracellular MnP seems to be a key enzyme in the conversion process.