Search

Basidiomycete white rot and litter-decomposing fungi secrete unspecific oxidative enzymes for degradation of aromatic polymer lignin. Recalcitrant organopollutants with structural similarities to lignin can also be degraded by these enzymes, i.e., laccases and class II heme peroxidases. Laccases belong to a superfamily of multicopper oxidases, which have been characterized in basidiomycete and ascomycete fungal species. Laccases catalyze one-electron transfer reactions from phenolic and low-redox-potential compounds with the concomitant reduction of molecular oxygen to water. In the presence of small molecular weight mediator compounds, the substrate spectrum of laccases expands to non-phenolic molecules and larger organic polymers. Fungal laccases have broad substrate range, and therefore they have attracted attention as “green catalysts” in different areas of biotechnology including bioremediation. Applications for fungal laccases are found in wastewater treatment, detoxification, and decolorization of industrial effluents as well as in bioremediation of contaminated soils. In this chapter, we describe the properties of fungal laccases, their reactions with mediator compounds, and their recombinant production in different host organisms. We also discuss the potential and challenges as well as the recent trends of the use of fungal laccases in bioremediation of wastewaters and soils contaminated with various xenobiotics.

To find out ability of fourteen basidiomycetes and four ascomycetes strains to grow in the presence of synthetic colour dyes and to degrade them, fungi were cultivated on the malt agar plates containing 0.5 g kg-1 dye, either Remazol Brilliant Blue R, Remazol Brilliant Yellow GL, Remazol Brilliant Orange 3 R, Reactive Blue 4, Remazol Brilliant Red F3B or Reactive Black 5. Fungi representing basidiomycetes were Phlebia radiata (FBCC 43), Tremella encephala (FBCC 1145), Dichomitus squalens (FBCC 312), Physisporinus rivulosus (syn. Obba rivulosa, FBCC 939), Cerrena unicolor (FBCC 387), Pleurotus abieticola (FBCC 517), Phanerochaete velutina (FBCC 941), Agrocybe praecox (FBCC 476), Trametes pubescens (FBCC 735), Pleurotus ostreatus (FBCC 498), Fomitopsis pinicola (FBCC 18), Postia placenta (= syn. Rhodonia placenta, FBCC 112), Gloeophyllum trabeum (FBCC 328) and Piptoporus betulinus (FBCC 1191). Ascomycetes belonged to genera Alternaria (HAMBI 3289), Epicoccum (HAMBI 3291), Fusarium (HAMBI 3292) or Chaetomium (HAMBI 3291). The growth rate of P. rivulosus belonged to three highest among the 14 tested basidiomycetes with five dyes, but not in the case of Remazol Brilliant Red F3B containing plates. The growth rate of A. praecox belonged to three lowest among the 14 tested basidiomycetes on Remazol Brilliant Blue R, Remazol Brilliant Yellow GL, Remazol Brilliant Red F3B, Reactive Black 5 and malt agar. The growth rate of Chaetomium sp. was the highest among the four ascomycetes on all tested plates. Decolorization was seen with 7 basidiomycetous strains on Remazol Brilliant Blue R, with 7 basidiomycetes on Remazol Brilliant Orange 3 R, with 8 basidiomycetes on Reactive Blue 4 and 11 basidiomycetes on Reactive Black 5 containing plates. T. encephala did not decolorize any of the tested six dyes. Epicoccum sp. and Chaetomium sp. decolorized Reactive Black 5 dye containing plates. None of the fourteen basidiomycetous or four ascomycetes were able to degrade all the tested six dyes.

Open Access article paid by the author Background: Wood rotting white-rot and litter-decomposing basidiomycetes form a huge reservoir of oxidative enzymes, needed for applications in the pulp and paper and textile industries and for bioremediation. Objective: The aim was (i) to achieve higher throughput in enzyme screening through miniaturization and automatization of the activity assays, and (ii) to discover fungi which produce efficient oxidoreductases for industrial purposes. Methods: Miniaturized activity assays mostly using dyes as substrate were carried out for lignin peroxidase, versatile peroxidase, manganese peroxidase and laccase. Methods were validated and 53 species of basidiomycetes were screened for lignin modifying enzymes when cultivated in liquid mineral, soy, peptone and solid state oat husk medium. Results: Manganese peroxidases were the most common enzymes produced by 96% of the species. They typically had acidic pH optima, although Hyphodontia sp., Pleurotus pulmonarius and Trametes ochracea produced enzymes highly active at pH 7. Versatile peroxidase was produced by 66% of the fungi with efficient production from Phlebia radiata, P. pulmonarius and Galerina marginata. Novel lignin peroxidase producing fungi Cylindrobasidium evolvens and Daedaleopsis septentrionalis were found among the 26% of the species showing here lignin peroxidase production. Laccase was shown in 92% of the species. Several fungi produced laccase active at pH 7, which is noteworthy because usually laccases of white-rot fungi are efficient and relevant for many industrial applications. Conclusion: Automated screening allowed us to monitor many specific enzyme activities and extend the range of assay conditions from relatively small fungal cultivation sample volumes.

Effects of Gadolinium and Tin to the Production of Oxidative Enzymes and the Growth of Five Basidiomycetous Fungi Effects of gadolinium (Gd) and tin (Sn) on the growth and production of oxidative enzymes with five basidiomycetous fungi were tested. For this study we have selected well-known white-rot fungi Obba rivulosa and Kuehneromyces mutabilis, in addition to this we have tested three new isolates, the white-rot fungus Phlebia subochracea, the litter-degrading fungus Gymnopus dryophilus and the brown-rot fungus Heliocybe sulcata. This approach allowed us to find possible new sources for oxidative enzymes, such as laccases and versatile peroxidases (VPs). All five tested fungi grew in the presence of Gd (0-200 mg/l) or Sn (0-200 mg/l) on ABTS (2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) containing plates. The growth rate of H. sulcata was tolerant to Gd and Sn (0- 200 mg/l). The growth rates of P. subochracea and G. dryophilus were sensitive to Gd (5-200 mg/l) and Sn (5-200 mg/l). O. rivulosa, K. mutabilis, P. subochracea and G. dryophilus formed colour zones on the ABTS plates indicating that these fungi produced oxidative enzymes, most probably laccases. The brown-rot fungus H. sulcata did not form colour zone on the ABTS plate indicating that this fungus did not produce laccase. The production of laccase with G. dryophilus and K. mutabilis was tolerant to Gd (0-200 mg/l) and Sn (0-200 mg/l). The production of laccase with P. subochracea was sensitive to Gd (5-200 mg/l) and Sn (5-200 mg/l). P. subochracea decolorized the dye Reactive Black 5 without or with Gd and Sn (0- 200 mg/l) indicating the production of VP. O. rivulosa, K. mutabilis, G. dryophilus and H. sulcata did not produce VP. The production of VP by P. subochracea was sensitive to 200 mg/l Gd and Sn.

Identification of bioactive components from complex natural product extracts can be a tedious process that aggravates the use of natural products in drug discovery campaigns.This study presents a new approach for screening antimicrobial potential of natural product extracts by employing a bioreporter assay amenable to HPLC-based activity profiling.A library of 116 crude extracts was prepared from fungal culture filtrates by liquid-liquid extraction with ethyl acetate, lyophilised, and screened against Escherichia coli using TLC bioautography. Active extracts were studied further with a broth microdilution assay, which was, however, too insensitive for identifying the active microfractions after HPLC separation. Therefore, an assay based on bioluminescent E. coli K-12 (pTetLux1) strain was coupled with HPLC micro-fractionation.Preliminary screening yielded six fungal extracts with potential antimicrobial activity. A crude extract from a culture filtrate of the wood-rotting fungus, Pycnoporus cinnabarinus (Jacq.) P. Karst. (Polyporaceae), was selected for evaluating the functionality of the bioreporter assay in HPLC-based activity profiling. In the bioreporter assay, the IC50 value for the crude extract was 0.10 mg/mL. By integrating the bioreporter assay with HPLC micro-fractionation, the antimicrobial activity was linked to LC-UV peak of a compound in the chromatogram of the extract. This compound was isolated and identified as a fungal pigment phlebiarubrone.HPLC-based activity profiling using the bioreporter-based approach is a valuable tool for identifying antimicrobial compound(s) from complex crude extracts, and offers improved sensitivity and speed compared with traditional antimicrobial assays, such as the turbidimetric measurement.

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.

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.

The lignin-degrading, biopulping white-rot fungus Physisporinus rivulosus secretes several laccases of distinct features such as thermostability, extremely low pH optima and thermal activation for oxidation of phenolic substrates. Here we describe the cloning, heterologous expression and structural and enzymatic characterisation of two previously undescribed P. rivulosus laccases. The laccase cDNAs were expressed in the methylotrophic yeast Pichia pastoris either with the native or with Saccharomyces cerevisiae α-factor signal peptide. The specific activity of rLac1 and rLac2 was 5 and 0.3 μkat/μg, respectively. However, mutation of the last amino acid in the rLac2 increased the specific laccase activity by over 50-fold. The recombinant rLac1 and rLac2 enzymes demonstrated low pH optima with both 2,6-dimethoxyphenol (2,6-DMP) and 2,2'-azino-bis(3-ethylbenzathiazoline-6-sulfonate). Both recombinant laccases showed moderate thermotolerance and thermal activation at +60 °C was detected with rLac1. By homology modelling, it was deduced that Lac1 and Lac2 enzymes demonstrate structural similarity with the Trametes versicolor and Trametes trogii laccase crystal structures. Comparison of the protein architecture at the reducing substrate-binding pocket near the T1-Cu site indicated the presence of five amino acid substitutions in the structural models of Lac1 and Lac2. These data add up to our previous reports on laccase production by P. rivulosus during biopulping and growth on Norway spruce. Heterologous expression of the novel Lac1 and Lac2 isoenzymes in P. pastoris enables the detailed study of their properties and the evaluation of their potential as oxidative biocatalysts for conversion of wood lignin, lignin-like compounds and soil-polluting xenobiotics.

A novel sampling method to evaluate extracellular fungal enzyme activities was developed and the validity tested for agar media. The method is based on centrifugation of small agar pieces taken from growing fungal solid-state cultures. Centrifuge tubes that allow spinning liquid out from small samples containing, for example, the hyphal front of a growing mycelium are essential for the protocol. Centrifugation recovers a liquid phase from the samples, which contains soluble material including many enzymes. The recovery of two added model enzymes, namely laccase and manganese peroxidase (MnP), from agar media was sufficient (ranging from 50 % to 75 %) but the addition of humic material into agar decreased the observed MnP activity significantly to approx. 25 % of the stock solution. Using growing cultures, the presence of humus as well as Scots pine sawdust on Hagem’s agar plates induced the production of laccase and peroxidase in certain fungi, which indicates that the method is suitable for screening enzyme activities on different growth media or with variable additives or growth conditions. The use of the presented sampling method for functional enzyme fingerprinting of different fungi may be a promising tool for investigating the behaviour and ecological role of forest soil fungi. This method also allows obtaining spatial data from very small and defined areas of solid fungal cultures, e.g. from microcosms.

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.

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.

The utility of simple syringyl-type phenolics of plant origin and some common synthetic compounds were compared with regard to their ability to act as laccase mediators in the oxidation of non-phenolic lignin model compounds. It was demonstrated that syringyl-type phenols, especially acetosyringone and methyl syringate, were able to mediate the oxidation of substrates of high oxidation potential by a laccase with a low redox potential. A mediator dose around 10% showed good performance compared to the equimolar quantities needed when the synthetic compounds 1-hydroxybenzotriazole or N-hydroxyacetanilide were used.

BACKGROUND: For most dioxin-contaminated sawmill soils, combustion is recommended. However, the process may be inefficient if the soil has a high organic matter content. The use of saprotrophic basidiomycetous fungi is an alternative for pretreatment of this kind of soil. A total of 147 fungi were evaluated for their ability to grow in sawmill soil. From this screening, the best soil colonizing fungi were selected to study their enzyme activities and degradation of soil organic matter. Pine (Pinus sylvestris) bark was used as a co-substrate to propagate the fungi into the soil. The activities of manganese peroxidase (MnP), laccase, endo-1,4-β-glucanase, endo-1,4-β-xylanase, and endo-1,4-β-mannanase were analysed from the inocula and fungal treated soil. RESULTS: The screening revealed that 56 out of 147 fungi were able to grow in non-sterile soil, and most of them were litter-decomposing fungi (LDF). In pine bark cultures, the highest enzyme activities were observed with Phanerochaete velutina, which produced 5 U g−1 of MnP. The activity of endo-1,4-β-glucanase was generally higher than that of other hydrolytic enzymes. The highest carbon loss from soil with a high organic matter content was achieved by P. velutina (3.4%) and Stropharia rugosoannulata (2.4%). CONCLUSIONS: Many LDF, and in addition the white-rot fungus P. velutina, are potential degraders of soil organic matter since they showed good growth and respiratory activity. Pine bark was a suitable lignocellulosic co-substrate and a good promoter of MnP activity. Copyright © 2009 Society of Chemical Industry

BACKGROUND: Combustion at high temperature is a common treatment method for heavily contaminated soils. The capacity of the combustion process is negatively correlated with organic matter content of the soil. Thus, by reducing the amount of soil organic matter, batch size could be increased and the combustion process improved. In this study, the possibility to pre-treat soil containing high levels of organic matter with white-rot and litter-decomposing fungi was examined and scaled up. RESULTS: Calculations based on the CO2 production in laboratory experiments indicated that 20% of the soil organic carbon would have been degraded in 6 months when treated with Sphaerobolus stellatus and 10% when treated with Stropharia rugosoannulata. In a pilot-scale experiment with S. rugosoannulata mass loss due to degradation of soil organic matter accounted for 10% of the total weight of the soil in 6 months. CONCLUSION: A fungal pre-treatment process for contaminated soils with high organic matter content was developed. Good results were obtained with S. stellatus and S. rugosoannulata and the process was successfully scaled up to 300 kg scale. Copyright © 2009 Society of Chemical Industry

Suggested applications of bio–ligninolytic systems in pulp and paper industry are usually based on selective removal or in situ modification of lignin. For example, it is possible using lignin–degrading white– rot fungi to decrease the amount of energy needed for mechanical pulping, i.e. biopulping, or to facilitate pulp bleaching (Kirk and Chang, 1990; Messner and Srebotnik, 1994; Ragauskas, 2002, Hatakka et al., 2002). Besides wood, annual plants, e.g. cereal straw and grass species contain substantial amounts of cellulose, which could be used as feed for ruminants, as a growth substrate for microorganisms and also as raw material for pulp and paper industry. However, due to the close association between cellulose and other plant cell wall polymers, i.e. lignin and hemicellulose (Eriksson et al., 1990; Daniel, 1994) cellulose is not easily available as a carbon or fiber source unless the structure of lignin is chemically or biologically modified or lignin is partially removed. Microbial delignification may be considered as a noteworthy alternative (Kirk and Farrell, 1987; Hatakka et al., 1989; Lewis and Yamamoto, 1990; Sarikaya and Ladish, 1997; Scott et al., 2002). In these cases a key question is how to break down the connection between lignin and carbohydrates, especially hemicellulose. The knowledge of the critical factors that are involved in the biodegradation of lignin carbohydrate complex (LCC) would help to understand and regulate selective delignification. The white–rot fungi that belong to Basidiomycetes are the most efficient of all known lignin degraders (Eriksson et al., 1990, Hatakka, 2001) but they also degrade cellulose and hemicellulose. Among these fungi Phlebia radiata is a very efficient degrader of lignin (Hatakka and Uusi–Rauva, 1983), it preferentially degrades lignin (Ander and Eriksson, 1977; Hatakka, 1983, 1994) but also was characterized as cellulase (Rogalski et al., 1993; Longa, 1997) and hemicellulase producer (Rogalski et al., 1993a; Tokarzewska– Zadora, 2001). Phlebia. radiata produces directly lignin–modifying enzymes, including lignin peroxidase (ligninase, LiP; EC 1.11.1.14), manganese peroxidase (MnP; EC 1.11.1.13), laccase (EC 1.10.3.2) and dioxygenase (EC 1.13.11), which have been characterized (Niku–Paavola et al., 1988; 1990; Hatakka et al., 1989; Karhunen et al., 1990; Lundell, 1993; Vares et al., 1995; Rogalski et al., 1996). The second group of enzymes involved in lignin degradation does not attack wood components directly. They include aryl alcohol oxidase and glyoxal oxidase providing H2O2 for the peroxidases, and also the feed–back type enzymes as glucose oxidase and cellobiose:quinone oxidoreductase (cellobiose dehydrogenase) that play an important role in joining the metabolic chains during the bioconversion of high– molecular mass wood constituents. All these enzymes Directional Degradation of Lignocellulose by Phlebia radiata

In this study we investigated the use of three industrial waste materials as substrates for white-rot fungi in solid-state cultivation (SSC). The materials used were oat husks and waste from paper process industry: fibre sludge and combined fibre and de-inking sludge (FDS). The aim of the work was to find suitable fungi able to grow and produce lignin modifying enzymes, manganese peroxidase (MnP) and laccase, on the used waste materials. Fifteen strains were tested. The best enzyme producing strains were Trametes versicolor K 120a2 and Cerrena unicolor T 71. C. unicolor T 71 showed the greatest potential for industrial use because of its ability to produce significant amounts of both enzymes simultaneously in short time period. The highest enzyme activities (MnP 64 nkat/g DW and laccase 178 nkat/g DW) were obtained on the medium containing sole oat husks or oat husks supplemented with 20% (w/w) of FDS.

Manufacturing of mechanical pulp is a very energy-consuming process. Application of enzymes to wood chips is an attractive alternative to decrease energy demand in the refining process and to introduce novel functional properties on fibers. A variety of enzymes were applied to plug screw compressed chips in order to improve enzyme access onto wood fibers. Consumption of refining energy was examined with a laboratory low-intensity refiner after 6-h enzyme treatments with manganese peroxidase (MnP), laccase-mediator system, pectinase, or a cellulase mixture. The results were compared to biopulping with the white-rot fungus Physisporinus rivulosus . Specific energy consumption resembling the first-stage refining was measured, and chemical modifications on the fibers were evaluated. The specific energy consumption in the refining of Scots pine wood chips treated with MnP decreased about 11% when compared to the untreated reference chips, and in the refining of Norway spruce somewhat less, 6%. Fungal pre-treatment resulted in similar energy savings on spruce as MnP treatment did. Hydrolytic enzyme and MnP treatments on pine resulted in similar energy savings on average, though the hydrolytic enzyme treatments at their best reached to about 15% energy savings. Polyelectrolyte titration indicated increased surface charge in the case of MnP treated pine pulps, which were refined to low freeness values (CSF 85–130 ml). Most laboratory handsheet properties, i.e. strength, light scattering and opacity, were improved at given specific refining energy. Only brightness was slightly decreased. The MnP treatment seems to be a promising concept for energy savings and fiber surface modifications.

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 (40 g Pb of kg ww soil −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 Halvala (37 g Pb of kg ww 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 (40 g Pb of kg ww soil −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 (40 g Pb of kg ww soil −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.

Demethoxylation reactions in the cultures of the brown-rot fungi Gloeophyllum trabeum and Poria placenta were studied by determining the evolution of (14)CO(2) from a non-phenolic lignin model, beta-O-4 dimer, [O(14)CH(3)]-labelled at position 4 in the A ring (model I), and from [O(14)CH(3)]-labelled vanillic acid (model II). The fungi were grown under oxygen or air atmosphere on an agar medium with or without spruce sapwood blocks. The dimeric model (I) was impregnated onto agar or wood block in cultures to clarify the possible effect of wood as growth substrate. In the case of vanillic acid (model II), birch wood was used. The effect of supplemented nutrient nitrogen (2 mM N) and glucose (0.1 or 1.0% w/v) on demethoxylation was also studied. G. trabeum enhanced the production of (14)CO(2) from the dimer in the presence of spruce wood blocks. It released (14)CO(2) from the methoxyl groups giving 30-60% of the applied activity in 8 weeks. P. placenta produced almost 30% (14)CO(2 )from vanillic acid (model II) in 9 weeks under oxygen, but from the methoxyl group of the dimer only 3% of (14)CO(2) was evolved in 4 weeks. The biomasses determined as ergosterol assay showed variation from 14 to 226 microg g(-1) dry weight of agar, and 2 to 9 microg g(-1 )of wood, but they did not correlate with the production of (14)CO(2). The results indicate that these brown-rot fungi possess different mechanisms for demethoxylation.

The white-rot basidiomycete Physisporinus rivulosus strain T241i is highly selective for degradation of softwood lignin, which makes this fungus suitable for biopulping. In order to promote laccase production, P. rivulosus was cultivated in nutrient-nitrogen sufficient liquid media containing either charcoal or spruce sawdust as supplements. Two laccases with distinct pI values, Lac-3.5 and Lac-4.8, were purified from peptone-spruce sawdust-charcoal cultures of P. rivulosus. Both laccases showed thermal stability at up to 60 degrees C. Lac-4.8 was thermally activated at 50 degrees C. Surprisingly, both laccases displayed atypically low pH optima (pH 3.0-3.5) in oxidation of the commonly used laccase substrates syringaldazine (4-hydroxy-3,5-dimethoxybenzaldehyde azine), 2,6-dimethoxyphenol and guaiacol (2-methoxyphenol). Steady-state kinetic measurements pointed to unusually low affinity to guaiacol at low pH, whereas the kinetic constants for the methoxyphenols and ABTS were within the ranges reported for other fungal laccases. The combination of thermotolerance with low pH optima for methoxylated phenol substrates suggests that the two P. rivulosus T241i laccases possess potential for use in biotechnological applications.

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.

The influence of aromatic phenolic and non-phenolic acids on manganese peroxidase (MnP)-dependent peroxidation of linoleic acid, and oxidation of a non-phenolic lignin model compound (LMC) was studied. Phenolic compounds inhibited both the MnP-dependent lipid peroxidation (LPO) and non-phenolic LMC degradation in the system. The antioxidant activity of the aromatic compounds in the enzymatic system with MnP-dependent LPO depends on the presence of the phenolic hydroxyl groups attached to the aromatic ring structure, the methoxylation of the hydroxyl group in the ortho position in diphenolics, and number of carbon atoms in the side chain. Natural phenolic compounds inhibit the oxidation of non-phenolic lignin in the system based on MnP-mediated LPO, but do not prevent it. This result indicates that MnP-mediated LPO may play an important role in lignin degradation even in the presence of the phenolic antioxidant compounds, and supports the possibility of the involvement of LPO in the degradation of lignin i...

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.

The biological conversion of plant lignocellulose plays an essential role not only in carbon cycling in terrestrial ecosystems but also is an important part of the production of second generation biofuels and biochemicals. The presence of the recalcitrant aromatic polymer lignin is one of the major obstacles in the biofuel/biochemical production process and therefore microbial degradation of lignin is receiving a great deal of attention. Fungi are the main degraders of plant biomass, and in particular the basidiomycete white rot fungi are of major importance in converting plant aromatics due to their ability to degrade lignin. However, the aromatic monomers that are released from lignin and other aromatic compounds of plant biomass are toxic for most fungi already at low levels, and therefore conversion of these compounds to less toxic metabolites is essential for fungi. Although the release of aromatic compounds from plant biomass by fungi has been studied extensively, relatively little attention has been given to the metabolic pathways that convert the resulting aromatic monomers. In this review we provide an overview of the aromatic components of plant biomass, and their release and conversion by fungi. Finally, we will summarize the applications of fungal systems related to plant aromatics.

The biological conversion of plant lignocellulose plays an essential role not only in carbon cycling in terrestrial ecosystems but also is an important part of the production of second generation biofuels and biochemicals. The presence of the recalcitrant aromatic polymer lignin is one of the major obstacles in the biofuel/biochemical production process and therefore microbial degradation of lignin is receiving a great deal of attention. Fungi are the main degraders of plant biomass, and in particular the basidiomycete white rot fungi are of major importance in converting plant aromatics due to their ability to degrade lignin. However, the aromatic monomers that are released from lignin and other aromatic compounds of plant biomass are toxic for most fungi already at low levels, and therefore conversion of these compounds to less toxic metabolites is essential for fungi. Although the release of aromatic compounds from plant biomass by fungi has been studied extensively, relatively little attention has been given to the metabolic pathways that convert the resulting aromatic monomers. In this review we provide an overview of the aromatic components of plant biomass, and their release and conversion by fungi. Finally, we will summarize the applications of fungal systems related to plant aromatics.

Manganese peroxidase (MnP) production in the white-rot basidiomycete Physisporinus rivulosus T241i was studied. Separate MnP isoforms were produced in carbon-limited liquid media supplemented with Mn(2+), veratryl alcohol, or sawdust. The isoforms had different pH ranges for the oxidation of Mn(2+) and 2,6-dimethoxyphenol. Although lignin degradation by white-rot fungi is often triggered by nitrogen depletion, MnPs of P. rivulosus were efficiently produced also in the presence of high-nutrient nitrogen, especially in cultures supplemented with veratryl alcohol. Two MnP encoding genes, mnpA and mnpB, were identified, and their corresponding cDNAs were characterized. Structurally, the genes showed marked dissimilarity, and the expression of the two genes implicated quantitative variation and differential regulation in response to manganese, veratryl alcohol, or sawdust. The variability in regulation and properties of the isoforms may widen the operating range for efficient lignin degradation by P. rivulosus.

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.

Linarin (acacetin-7-O-beta-d-rutinoside) from the flower extract of Mentha arvensis showed selective dose dependent inhibitory effect on acetylcholinesterase.

Laccases are phenol-oxidizing, multicopper enzymes produced by fungi, plants, insects and bacteria. Fungal laccases are involved in ecologically important processes such as decomposition of lignocellulose (wood and plant material). In this work, in order to find out the role of fungal laccases upon wood colonisation and lignin decay, we describe expression of laccase-encoding genes in the white rot basidiomycete Phlebia radiata 79, when the fungus grows on its natural substrates, that is on softwood (Alnus incana) and hardwood (Picea abies). Clones for two laccase-encoding genes, the previously described Pr-lac1 and a new gene Pr-lac2 were characterized. Pr-lac2 coding region is interrupted by 12 introns and the deduced Lac2 protein displays a higher pI value (5.8) than Lac1 (pI 3.2-3.5). Phylogenetic analysis indicates differential evolution for the two laccases, and Lac2 demonstrates the highest sequence identity with Trametes laccases (66%). Transcripts of Pr-lac1 were the most abundant both in solid-state softwood and semi-solid hardwood cultures, as analyzed by competitive RT-PCR and Northern hybridization. On spruce wood chips, Pr-lac1 and Pr-lac2 were expressed within 2-3 weeks of growth together with manganese and lignin peroxidase-encoding genes. Our results indicate wood-promoted but time-dependent regulation of expression for the two, at protein and gene level distinct P. radiata laccases.

Extracellular laccases from submerged cultures of Coriolus versicolor BKM F-116, Panus tigrinus 8/18, Phlebia radiata 79 (ATCC 64658), Phlebia tremellosa 77-51 and from cultures of Pa. tigrinus 8/18, Ph. radiata 79 and Agaricus bisporus D-649 grown on wheat straw (solid-state fermentation) were purified. All enzymes from submerged cultures had a blue colour and characteristic absorption and EPR spectra. Laccases from the solid-state cultures were yellow-brown and had no typical blue oxidase spectra and also showed atypical EPR spectra. Comparison of N-terminal amino acid sequences of purified laccases showed high homology between blue and yellow-brown laccase forms. Formation of yellow laccases as a result of binding of lignin-derived molecules by enzyme protein is proposed.

Paecilomyces inflatus isolated from municipal waste compost was found to have cellulolytic activity in several solid and liquid media. This study was done to reveal the multifarious effects of municipal waste compost on endoglucanase activity of P. inflatus. The highest enzyme activities under the conditions of solid-state fermentation were measured in authentic compost samples compared with wood, straw and bran substrates. In surface liquid cultures glucose, cellobiose, xylan, Avicel cellulose, carboxymethylcellulose (CM-cellulose), starch and citrus pectin were used as carbon sources. All carbon sources supported the growth of P. inflatus. However, only CM-cellulose, cellobiose and pectin noticeably stimulated endoglucanase (EG) activity. Further stimulation of EG activity was obtained in cultures containing 1% CM-cellulose as a carbon source by supplementation with low-molecular mass aromatic compounds vanillin, veratric acid and benzoic acid, and with soil humic acid (SHA). SHA and veratric acid were found to be the most efficient elicitors of the cellulolytic activity. P. inflatus was able to utilize nitrate and ammonium as pure nitrogen sources in media containing cellulose.

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 \({}^{{14}}{\text{C}}_{{\text{ $ \beta $ }}} \)-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 (

Two new, at primary sequence and protein structure levels different, manganese peroxidase encoding genes from the white rot basidiomycete Phlebia radiata are described. Both genes are expressed in liquid cultures of P. radiata containing milled alder wood or glucose as carbon source, and high Mn2+ concentration. The gene Pr-mnp2 contains 7 introns and codes for a 390 amino-acid polypeptide, whereas Pr-mnp3 presents 11 introns and codes for a 362 amino-acid protein. The 3-D molecular models confirm this diversity; the predicted Pr-MnP2 with a long C-terminal extension has the highest structural similarity with the crystal structure of Phanerochaete chrysosporium MnP1, whereas the shorter Pr-MnP3 protein is structurally more related to lignin peroxidases (P. chrysosporium LiPH8/H2). In Pr-MnP3, however, an alanine replaces the exposed tryptophan present in LiP and versatile peroxidases, and both Pr-MnPs include the conserved Mn2+-binding amino-acid ligands. This is the first occasion when two enzymes of similar function and origin fall into phylogenetically distinct subfamilies within the expanding dendrogram of the class II fungal secretory heme peroxidases.

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.

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 (28 μmol/g dry wood) and a high MnP activity (24 nkat/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 p I values between 3.4 and 3.9 and molecular masses between 47 and 52 kDa. The four laccase isoforms had p I values between 3.1 and 3.3 and molecular masses between 66 and 68 kDa. 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.

The main manganese peroxidase (MnP) isoenzyme of Agaricus bisporus ATCC 62459 produced in lignocellulose-containing cultures was isolated, cloned and sequenced. In liquid medium, where MnP was previously detected only in trace amounts, the production of MnP was enhanced by rye and wheat bran supplements. The pI (3.25) and N-terminal amino acid sequence (25 aa) of the enzyme from bran-containing cultures were identical to those reported from compost-isolated MnP1. MnP1 is a 328-aa long polypeptide preceded by a 26-aa leader peptide. The nucleotide sequence and putative amino acid sequence of MnP1 reveal its similarity to Pleurotus ostreatus MnP3 (62.5%), Lepista irina versatile peroxidase (VP) (61.8%) and Pleurotus eryngii VPs VPL2 and VPL1 (61.9% and 61.2%, respectively). The intron-exon structure resembles that of P. ostreatus MnP1 and P. eryngii VPL1. Despite the sequence similarity to VPs, in the A. bisporus MnP1 sequence, alanine (A163) is present instead of tryptophane (W164), distinguishing it from the veratryl alcohol oxidising P. eryngii VPLs. The MnP sequence can be used as a tool to examine the pattern of ligninolytic gene expression during the growth and fruiting of A. bisporus to optimise compost composition, fungal growth and mushroom production.

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.

In this study, we show that the wild-type strain Phanerochaete chrysosporium ME-446 is able to effectively produce manganese peroxidase (MnP) and lignin peroxidase (LiP) in submerged cultures under nitrogen and carbon non-limiting conditions, if the lignocellulosic substrate is added to the medium. No activity of LiP or MnP could be detected when the fungus was grown in the basal glucose–peptone–corn steep liquor medium. Only the addition of the lignocellulose-containing substrates, wheat straw or hemp woody core, to the medium led to production of the enzymes. This result strongly indicates that some compounds derived from the lignocellulosic substrates may function as inducers for production of the ligninolytic peroxidases. In the presence of the lignocellulosic substrates high amounts of organic nitrogen source (peptone; 3–4 g l−1) did not repress production of the ligninolytic peroxidases, as it commonly occurs with the wild-type strains of P. chrysosporium, but instead stimulated it. A simple medium, which does not require addition of Tween 80 or oxygen purging, was developed for effective production of ligninolytic peroxidases by P. chrysosporium ME-446 in submerged culture. The medium allows simultaneous production of high activities of both MnP and LiP.

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.

We investigated the degradation of synthetic lignin (14C-ring-labeled DHP) by various white-rot fungi in soil. Soil contained lignocellulose substrate (wheat straw), and the mixture was autoclaved before inoculation. The fungal species were Abortiporus biennis, Bjerkandera adusta, Dichomitus squalens, Phanerochaete chrysosporium, Phanerochaete sordida, Phlebia radiata, Pleurotus ostreatus, Trametes hirsuta, and Trametes versicolor. All fungi were incubated for 56 days with a straw-to-soil ratio of 1:5, and T. hirsuta was also cultured on straw without soil. In addition, B. adusta and T. hirsuta were studied with three other straw-to-soil ratios (1:2.5; 1:10; and 1:25). 14CO2 was collected during incubation, and after incubation cultures were successively extracted with water, dioxane, and alkali, and the residue was combusted. Mineralization of 14C-DHP by fungal inocula in soil was 4% with P. sordida and 23% with T. versicolor. Without soil T. hirsuta mineralized 30% of 14C-DHP, while the mineralization in autoclaved controls was only 0.2%. As the amount of straw decreased, fungi grew less readily, and T. hirsuta mineralized smaller amounts of 14C-DHP to 14CO2. However, the mineralization by B. adusta did not alter significantly until the straw-to-soil ratio was as low as 1:25. The dioxane fraction, representing unmodified 14C-DHP, decreased significantly during incubation of fungi with all straw treatments. By day 0, 62–64% of 14C-DHP was already bound to humic substances without fungal activity, but the mass balance of radiolabel in controls remained unchanged in spite of the incubation. White-rot fungi were apparently able to degrade the humic bound lignin, because the amount of radiolabel in humic fractions decreased in the course of incubation. However, most of 14C-DHP remained bound to humic substances, and the binding increased as the amount of straw decreased.

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.

The development of new biodegradable packaging materials, especially biodegradable plastics, has created a need for biodegradability testing. The European standard for controlled composting test was used in this study for assessing if the addition of a test material results in excess CO2 production in compost. This effect, designated as the priming effect, would give an erroneous result for biodegradation, which is based on CO2 formation from the test material. Glucose was selected as a test substrate because it is the degradation product of starch and cellulose, which are major compounds of many packaging materials. Both 14 C-glucose and non-labelled glucose was applied to nine compost samples of variable stability and age from two weeks to 1.5 years. CO2 and 14 CO2 evolution were measured during the incubation. Biodegradation of glucose in unstable composts (age ≤6 months) was negative and 14 CO2 evolution was poor, although the respective composts without glucose produced relatively high amounts of CO2. It was concluded that a negative priming effect was observed in unstable composts, in which glucose remained mostly non-degraded and apparently inhibited the mineralization of native organic matter in the compost. In stable composts (age ≥6 months), biodegradation of glucose was high and approximately equal to 14 C-glucose mineralization, i.e., the composts showed no priming effect. Young composts were unsuitable for controlled composting test due to lack of stability. It is important to ensure that the compost inoculum used for the test is sufficiently stable.

In this review, the present knowledge on the occurrence of cellulases, with a special emphasis on the presence of carbohydrate-binding modules (CBMs) in various fungal strains, has been summarized. The importance of efficient fungal cellulases is growing due to their potential uses in biorefinery processes where lignocellulosic biomasses are converted to platform sugars and further to biofuels and chemicals. Most secreted cellulases studied in detail have a bimodular structure containing an active core domain attached to a CBM. CBMs are traditionally been considered as essential parts in cellulases, especially in cellobiohydrolases. However, presently available genome data indicate that many cellulases lack the binding domains in cellulose-degrading organisms. Recent data also demonstrate that CBMs are not necessary for the action of cellulases and they solely increase the concentration of enzymes on the substrate surfaces. On the other hand, in practical industrial processes where high substrate concentrations with low amounts of water are employed, the enzymes have been shown to act equally efficiently with and without CBM. Furthermore, available kinetic data show that enzymes without CBMs can desorb more readily from the often lignaceous substrates, that is, they are not stuck on the substrates and are thus available for new actions. In this review, the available data on the natural habitats of different wood-degrading organisms (with emphasis on the amount of water present during wood degradation) and occurrence of cellulose-binding domains in their genome have been assessed in order to identify evolutionary advantages for the development of CBM-less cellulases in nature.

Fungal oxidative enzymes, such as peroxidases and laccases, are the key catalysts in lignin biodegradation in vivo, and consequently provide an important source for industrial ligninolytic biocatalysts. Recently, it has been shown that some syringyl-type phenolics have potential as industrial co-oxidants or mediators, in laccase-catalyzed modification of lignocellulosic material. We have now studied the effect of such mediators with ligninolytic peroxidases on oxidation of the most recalcitrant lignin model compounds. We found that they are able to enhance the manganese peroxidase (MnP) catalyzed oxidation reactions of small non-phenolic compounds, veratryl alcohol and veratrylglycerol β-guaiacyl ether (adlerol), which are not usually oxidized by manganese peroxidases alone. In these experiments we compared two peroxidases from white-rot fungi, MnP from Phlebia sp. Nf b19 and versatile peroxidase (VP) from Bjerkandera adusta under two oxidation conditions: (i) the Mn(III) initiated mediated oxidation by syringyl compounds and (ii) the system involving MnP-dependent lipid peroxidation, both with production of (hydrogen) peroxides in situ to maintain the peroxidase catalytic cycle. It was found that both peroxidases produced α-carbonyl oxidation product of veratryl alcohol in clearly higher yields in reactions mediated by phenoxy radicals than in lipid-peroxyl radical system. The oxidation of adlerol, on the other hand, was more efficient in lipid-peroxidation-system. VP was more efficient than MnP in the oxidation of veratryl alcohol and showed its lignin peroxidase type activity in the reaction conditions indicated by some cleavage of Cα–Cβ-bond of adlerol. Finally, the mediator assisted oxidation conditions were applied in the oxidation of synthetic lignin (DHP) and the structural analysis of the oxidized polymers showed clear modifications in the polymer outcome, e.g. the oxidation resulted in reduced amount of aliphatic hydroxyls indicated by 31P NMR.

Lignin-modifying enzymes have various promising applications such as biobleaching, biopulping, the functionalization of lignocellulosic materials, the modification of wood fibers, the remediation of contaminated soil and effluents, as well as improvement of the enzymatic hydrolysis of lignocellulosic substrates. In this study, the production of laccase and manganese peroxidase (MnP) in solid-state cultivation was examined. Oat husks were used as an inexpensive substrate for the white-rot fungus Cerrena unicolor PM170798 (FBCC 387). The addition of a fines fraction (consisting of oat flour and finely ground husks) enhanced MnP production fivefold and laccase production almost threefold. The enzyme production was studied first on a 100 g scale, and the cultivation experiments were then repeated at a larger laboratory-scale (4 kg) in a solid-state bioreactor. High enzyme activity levels were obtained (MnP: 340 nkat g(-1) DM, laccase: 470 nkat g(-1) DM). In addition, the correlation between the CO2 evolution rate and enzyme production was mathematically modeled from the bioreactor experimental data. The model parameters could be used to predict enzyme production.

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.

In this review, the present knowledge on the occurrence of cellulases, with a special emphasis on the presence of carbohydrate-binding modules (CBMs) in various fungal strains, has been summarized. The importance of efficient fungal cellulases is growing due to their potential uses in biorefinery processes where lignocellulosic biomasses are converted to platform sugars and further to biofuels and chemicals. Most secreted cellulases studied in detail have a bimodular structure containing an active core domain attached to a CBM. CBMs are traditionally been considered as essential parts in cellulases, especially in cellobiohydrolases. However, presently available genome data indicate that many cellulases lack the binding domains in cellulose-degrading organisms. Recent data also demonstrate that CBMs are not necessary for the action of cellulases and they solely increase the concentration of enzymes on the substrate surfaces. On the other hand, in practical industrial processes where high substrate concentrations with low amounts of water are employed, the enzymes have been shown to act equally efficiently with and without CBM. Furthermore, available kinetic data show that enzymes without CBMs can desorb more readily from the often lignaceous substrates, that is, they are not stuck on the substrates and are thus available for new actions. In this review, the available data on the natural habitats of different wood-degrading organisms (with emphasis on the amount of water present during wood degradation) and occurrence of cellulose-binding domains in their genome have been assessed in order to identify evolutionary advantages for the development of CBM-less cellulases in nature.