Search

The aim of this work was to investigate the poorly understood effects of co-culturing of two white rot fungi on the production of lignin-degrading enzyme activities. Four species, Ceriporiopsis subvermispora, Physisporinus rivulosus, Phanerochaete chrysosporium and Pleurotus ostreatus were cultured in pairs to study the degradation of aspen wood and the production of lignin-degrading enzymes. Potential of co-culturing for biopulping was evaluated. Chemical analysis of decayed aspen wood blocks showed that co-culturing of C. Subvermispora with P. Ostreatus could significantly stimulate wood decay, when compared to monocultures. Based on the fungi tested here, however, this effect is species-specific. Other combinations of fungi were slightly stimulating or not stimulatory. The pattern of lignin degradation was altered towards the acid insoluble part of lignin especially in co-cultures where P. Ostreatus was included as a partner. The use of agar plates containing the polymeric dye Poly R-478 showed elevated dye decolourization at the confrontation zone between mycelia. Laccase was significantly stimulated only in the co-culture of P. Ostreatus with C. Subvermispora. Manganese peroxidase activity was stimulated in co-cultures of P. Ostreatus with C. Subvermispora or with P. Rivulosus. Immunoblotting indicated changes in lignin-degrading enzymes and/or their isoform composition in response to co-culturing. This is the first report on the effects of co-culturing of potential biopulping fungi on wood degradation, and gives basic knowledge on fungal interactions during wood decay that can be utilized in practical applications. [Copyright &y& Elsevier]

Abstract: Production of the oxidoreductive lignin-modifying enzymes – lignin and manganese peroxidases (MnPs), and laccase – of the white-rot basidiomycete Phlebia radiata was investigated in semi-solid cultures supplemented with milled grey alder or Norway spruce and charcoal. Concentrations of nutrient nitrogen and Cu-supplement varied also in the cultures. According to extracellular activities, production of both lignin peroxidase (LiP) and MnP was significantly promoted with wood as carbon source, with milled alder (MA) and low nitrogen (LN) resulting with the maximal LiP activities (550 nkat l−1) and noticeable levels of MnP (3 μkat l−1). Activities of LiP and MnP were also elevated on high nitrogen (HN) complex medium when supplemented with spruce and charcoal. Maximal laccase activities (22 and 29 μkat l−1) were obtained in extra high nitrogen (eHN) containing defined and complex media supplemented with 1.5 mM Cu2+. However, the nitrogen source, either peptone or ammonium nitrate and asparagine, caused no stimulation on laccase production without Cu-supplement. This is also the first report to demonstrate a new, on high Cu2+ amended medium produced extracellular laccase of P. radiata with pI value of 4.9, thereby complementing our previous findings on gene expression, and cloning of a second laccase of this fungus. [Copyright &y& Elsevier]

Abstract: Unsaturated fatty acids have been proposed to mediate the oxidation of recalcitrant, non-phenolic lignin structures by fungal manganese peroxidases (MnP), but their precise role remains unknown. We investigated the oxidizability of three fatty acids with varying degrees of polyunsaturation (linoleic, linolenic, and arachidonic acids) by measuring conjugated dienes formation when lipid peroxidation was initiated either by MnP in the presence of Mn(II) or by chelated Mn(III). An inverse relationship between the degree of fatty acid unsaturation and the rate of peroxidation was found in both cases, but we also noted some differences between the two types of reaction. With MnP/Mn(II), the reaction developed slowly and resulted in sustained lipid peroxidation as determined by the formation of late-stage fatty acid degradation products. By contrast, the reaction with chelated Mn(III) was very rapid and did not result in the formation of these late-stage products, which suggests that this system failed to propagate the sustained radical chain reaction that is characteristic of complete lipid peroxidation. All three polyunsaturated fatty acids supported the co-oxidation of a non-phenolic lignin model compound by MnP, again showing an inverse relationship between the degree of unsaturation and reactivity, but chelated Mn(III) by itself supported only very low levels of fatty acid-mediated lignin model oxidation. These parallels in fatty acid reactivity are consistent with a reaction scheme in which Mn(III) acts as the proximal oxidant that initiates lipid peroxidation by MnP, thus generating fatty acid-derived radicals which in turn oxidize lignin structures. However, the results also suggest that the initial peroxyl radicals formed may not be the ligninolytic oxidants in this system. Instead, other radical oxidants produced during late-stage reactions of lipid peroxidation may be required. [Copyright &y& Elsevier]

Abstract: Ergosterol contents of six wood-rotting basidiomycetes were analyzed under different cultivation conditions. Four white-rot and two brown-rot fungi were cultivated in liquid synthetic medium with low nutrient nitrogen (2mM) and 0.1% glucose, and ergosterol in mycelial biomasses were measured weekly for 35 days. The highest ergosterol content per fungal dry mass in the white-rot fungi was found in Phanerochaete chrysosporium being 2100μgg−1, while in Ceriporiopsis subvermispora it was 1700μgg−1, Phlebia radiata 700μgg−1, and Physisporinus rivulosus 560μgg−1. In brown-rot fungi the ergosterol content was in Poria placenta 2868μgg−1 and in Gloeophyllum trabeum 3915μgg−1. On agar media, P. chrysosporium and P. radiata reached the highest ergosterol value in 7 days, while in wood block cultures the ergosterol contents were quite stable. The conversion factors for ergosterol-to-fungal biomass varied from 48 and 243, which were lower than values for ascomycetous soil fungi reported in the literature. [Copyright &y& Elsevier]

The impact of Pb contamination was tested to five hydrolytic (β-glucosidase, β-xylosidase, β-cellobiosidase, α-glucosidase and sulphatase) and two ligninolytic (manganese peroxidase, MnP and laccase) enzyme activities in the humus layer in the forest soil. The ability of eight selected litter-degrading fungi to grow and produce extracellular enzymes in the heavily Pb (40g Pb of kgwwsoil−1) contaminated and non-contaminated soil in the non-sterile conditions was also studied. The Pb content in the test soil was close to that of the shooting range at Hälvälä (37g Pb of kgww soil−1) in Southern Finland. The fungi were Agaricus bisporus, Agrocybe praecox, Gymnopus peronatus, Gymnopilus sapineus, Mycena galericulata, Gymnopilus luteofolius, Stropharia aeruginosa and Stropharia rugosoannulata. The Pb contamination (40g Pb of kgwwsoil−1) was deleterious to all five studied hydrolytic enzyme activities after five weeks of incubation. All five hydrolytic enzyme activities were significantly higher in the soil than in the extract of the soil indicating that a considerable part of enzymes were particle bound in the soils. Hydrolytic enzyme activities were higher in the non-contaminated soil than in the Pb contaminated soil. Fungal inocula increased the hydrolytic enzyme activities β-cellobiosidase and β-glucosidase in non-contaminated soils. All five hydrolytic enzyme activities were similar with fungi and without fungi in the Pb contaminated soil. This was in line that Pb contamination (40g Pb of kgwwsoil−1) depressed the growth of all fungi compared to those grown without Pb in the soil. Laccase and MnP activities were low in both Pb contaminated and non-contaminated soil cultures. MnP activities were higher in soil cultures containing Pb than without Pb. Our results showed that Pb in the shooting ranges decreased fungal growth and microbial functioning in the soil. [Copyright &y& Elsevier]

To find out microbial metabolic functioning and toxicity in a former sawmill area, carbon dioxide evolution, methane oxidation potential, 10 hydrolytic enzyme activities, Vibrio fischeri test, fluoresceine diacetate hydrolysis activity (FDA), soil pH, carbon, nitrogen and pentachlorophenol (PCP) content were measured at four sites. The area is contaminated with aged chlorophenols. Chlorophenol content of soil was analyzed with a novel HPLC-MS technique, which allowed to measure chlorophenols without derivatization. The sites had a pollution gradient from 0.5 to 15μg PCP g dw of soil−1. Endogenous carbon dioxide evolution, methane oxidation potential, butyrate-esterase, acetate-esterase, sulphatase and aminopeptidase activities were lower at the site 2 than 3, although the site 2 and 3 had similar content of carbon and nitrogen. The soil was toxic in V. fischeri test at the site 2, which had high content of PCP (3.93±1.00μg PCP g dw of soil−1). The results indicated that endogenous carbon dioxide evolution, methane oxidation potential, butyrate-esterase, acetate-esterase, sulphatase and aminopeptidase activities were sensitive to PCP in the soil. The results indicated that α-glucosidase, β-glucosidase, β-xylosidase, β-cellobiosidase, phosphomonoesterase, N-acetyl-glucosaminidase activity and FDA hydrolysis activity were not sensitive to PCP in the soil. Soil processes involved in the cycling of carbon, nitrogen, sulphur and phosphorus were only slightly vulnerable in the former sawmill area and most sensitive microbial species were probably replaced with more tolerant ones to maintain and recover functioning of the former sawmill soils. [Copyright &y& Elsevier]

Abstract: Linarin (acacetin-7-O-β-d-rutinoside) from the flower extract of Mentha arvensis showed selective dose dependent inhibitory effect on acetylcholinesterase. [Copyright &y& Elsevier]

Extracellular manganese peroxidase (MnP) was purified from liquid cultures of the litter-decomposing basidiomycetes Agrocybe praecox and Stropharia coronilla. Both fungi produced MnP increasingly in response to Mn2+ in the medium. A. praecox secreted two MnP isoforms with similar isoelectric points (pI) of 6.3–7.0 and a molecular weight (MW) of 42 kDa. MnP activity was not observed in Mn2+-free cultures of A. praecox. In Mn2+-supplemented cultures, S. coronilla produced at least two MnPs, of which the main isoform MnP1 has a pI of 6.3–7.1 and a MW of 41 kDa. In addition, S. coronilla possesses a partly constitutive MnP (MnP2) which was also detectable in Mn2+-free cultures, although its amount was considerably lower. MnP2 showed two distinct bands with acidic pIs of 3.5 and 3.7 in the IEF gel and has a MW of 41 kDa. There are indications for the existence of a third, likewise Mn2+-inducible enzyme (MnP3), that could not be separated from MnP2 but formed an additional band in eletrophoretic analyses (pI 5.1, MW 43 kDa). [Copyright &y& Elsevier]

Intracellular oxalate decarboxylase (ODC, EC 4.1.1.2) activity was screened in the mycelium of 12 white rot fungi. ODC activity was detected in the mycelial extracts of Dichomitus squalens, Phanerochaete sanguinea, Trametes ochracea, and Trametes versicolor (strain R/7) after addition of 5-mM oxalic acid to the liquid culture medium. In D. squalens, intracellular ODC activity increased six-fold with addition of oxalic acid. Production of extracellular organic acids by the four ODC-positive fungi was followed in liquid cultures and in solid state cultures of spruce wood chips by using HPLC and capillary zone electrophoresis (CZE). The four ODC-positive fungi secreted oxalic acid both in liquid and solid state cultures showing different production patterns until the end of growth (31 days). Upon cultivation on solid spruce wood chips, manganese peroxidase (MnP) activity peaked simultaneously in these fungi with the accumulation of extracellular oxalic acid. In addition to oxalic acid, glyoxylic and formic acids were detected in the cultures of D. squalens. [Copyright &y& Elsevier]

Abstract: The peroxidation of C18 unsaturated fatty acids by fungal manganese peroxidase (MnP)/Mn(II) and by chelated Mn(III) was studied with application of three different methods: by monitoring oxygen consumption, by measuring conjugated dienes and by thiobarbituric acid-reactive substances (TBARS) formation. All tested polyunsaturated fatty acids (PUFAs) were oxidized by MnP in the presence of Mn(II) ions but the rate of their oxidation was not directly related to degree of their unsaturation. As it has been shown by monitoring oxygen consumption and conjugated dienes formation the linoleic acid was the most easily oxidizable fatty acid for MnP/Mn(II) and chelated Mn(III). However, when the lipid peroxidation (LPO) activity was monitored by TBARS formation the linolenic acid gave the highest results. High accumulation of TBARS was also recorded during peroxidation of linoleic acid initiated by MnP/Mn(II). Action of Mn(III)-tartrate on the PUFAs mimics action of MnP in the presence of Mn(II) indicating that Mn(III) ions are involved in LPO initiation. Although in our experiments Mn(III) tartrate gave faster than MnP/Mn(II) initial oxidation of the unsaturated fatty acids with consumption of O2 and formation of conjugated dienes the process was not productive and did not support further development of LPO. The higher effectiveness of MnP/Mn(II)-initiated LPO system depends on the turnover of manganese provided by MnP. It is proposed that the oxygen consumption assay is the best express method for evaluation of MnP- and Mn(III)-initiated peroxidation of C18 unsaturated fatty acids. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: The composition of Scots pine bark, its degradation, and the production of hydrolytic and ligninolytic enzymes were evaluated during 90days 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. [Copyright &y& Elsevier]

This paper describes for the first time in detail the lignocellulose degradation system in Paecilomyces inflatus. The fungal genus Paecilomyces contributes the carbon turnover from lignin and carbohydrate plant residues, particularly in compost and soil environment, where basidiomycetes appear very seldom. We studied three different strains of P. inflatus, obtained from different ecophysiological and geographical origin. Various cultivation conditions were employed, and the chemical analysis of decayed straw, compost, birch and spruce wood chips indicated variable responses. Endoglucanase, xylanase and laccase were assayed. All strains of P. inflatus, regardless of their origin, altered the ambient pH in a similar manner in all investigated substrates, suggesting that all P. inflatus isolates may share the common regulatory system to control their environmental pH. The variability among strains of P. inflatus in their ability to remove lignocellulose components often was related to the nature of the substrate and the production of specific enzymes although it was not strictly correlated. This may implicate that other enzymes and/or even other parameters needed for lignocellulosics degradation in P. inflatus should be evaluated. Indications for specific adaptation strategies that may operate in P. inflatus were found. [Copyright &y& Elsevier]

Abstract: The white-rot fungus Physisporinus rivulosus T241i produced manganese peroxidase (MnP), laccase, and oxalic acid when it was grown on spruce (Picea abies) wood chips. This white-rot basidiomycete degrades lignin selectively and is promising for use in biopulping of softwood. During solid-state cultivation on wood chips the fungus produced oxalic acid (28μmol/g dry wood) and a high MnP activity (24nkat/g dry wood), while measured laccase activity was negligible. The prepurified laccase and MnP isoforms were fractionated with anion exchange chromatography. Characterization of the isolated MnP fractions indicated the presence of several MnP isoforms with pI values between 3.4 and 3.9 and molecular masses between 47 and 52kDa. The four laccase isoforms had pI values between 3.1 and 3.3 and molecular masses between 66 and 68kDa. The MnP and laccase isoform profiles did not vary during the 4-week cultivation. The N-terminal amino acid sequences of four laccase and two MnP isoenzymes resembled those of the two other selective white-rot fungi applied in biopulping, i.e. Ceriporiopsis subvermispora CZ-3 and the strain IZU-154. [Copyright &y& Elsevier]

Pre-treatment of wood chips with white-rot fungi (i.e. biopulping) has previously been shown to decrease the energy consumption in mechanical pulping. In this study, promising novel fungal isolates were found for biopulping of Norway spruce (Picea abies). Three hundred fungal isolates were evaluated by agar plate tests. According to the results, 86 isolates were chosen to a wood block decay test. The characteristics of the fungal attack and the wood decay were studied and chemical analyses of cellulose, lignin, and hemicelluloses in the residual wood were performed. The results showed both interspecies and intraspecies variations in the degradation patterns of these compounds. After a 10-week incubation period, 17 of the novel white-rot strains showed selective lignin degrading ability, based on the chemical analyses. Some of these lignin-selective strains also grew well even at +37 °C. The selectivity value, i.e. the lignin/cellulose loss ratio, was higher for 11 of the studied strains compared to the widely studied Ceriporiopsis subvermispora. In this study, Physisporinus rivulosus T241i was shown to be a superior novel isolate for softwood biopulping applications based on its growth characteristics through a wide temperature range and its selectivity towards lignin. [Copyright &y& Elsevier]