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Keywords:

  • white-rot;
  • soft-rot;
  • peroxidase;
  • peroxygenase;
  • Klason lignin

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The degradation of lignocellulose and the secretion of extracellular oxidoreductases were investigated in beech-wood (Fagus sylvatica) microcosms using 11 representative fungi of four different ecophysiological and taxonomic groups causing: (1) classic white rot of wood (e.g. Phlebia radiata), (2) ‘nonspecific’ wood rot (e.g. Agrocybe aegerita), (3) white rot of leaf litter (Stropharia rugosoannulata) or (4) soft rot of wood (e.g. Xylaria polymorpha). All strong white rotters produced manganese-oxidizing peroxidases as the key enzymes of ligninolysis (75–2200 mU g−1), whereas lignin peroxidase activity was not detectable in the wood extracts. Interestingly, activities of two recently discovered peroxidases – aromatic peroxygenase and a manganese-independent peroxidase of the DyP-type – were detected in the culture extracts of A. aegerita (up to 125 mU g−1) and Auricularia auricula-judae (up to 400 mU g−1), respectively. The activity of classic peroxidases correlated to some extent with the removal of wood components (e.g. Klason lignin) and the release of small water-soluble fragments (0.5–1.0 kDa) characterized by aromatic constituents. In contrast, laccase activity correlated with the formation of high-molecular mass fragments (30–200 kDa). The differences observed in the degradation patterns allow to distinguish the rot types caused by basidiomycetes and ascomycetes and may be suitable for following the effects of oxidative key enzymes (ligninolytic peroxidases vs. laccases, role of novel peroxidases) during wood decay.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The microbial decomposition and recycling of persistent natural polymers such as lignin and plant cell-wall polysaccharides (cellulose, hemicellulose, pectin) plays a key role in the global carbon cycle and has become relevant for the development of innovative biotechnological processes in different industries (pulp and paper, textile, chemical synthesis; Messner et al., 1998). The biological removal of encrusted lignin from the cell-wall complex in wood and nonwoody lignocelluloses is enzymatically difficult to accomplish and obviously a specific feature of filamentous fungi found in the phyla of Basidiomycota and Ascomycota, forming together the large subkingdom of Dikarya (‘higher fungi’). Ecophysiologically, the saprobes among them can be divided roughly into five types: white-rot, brown-rot and soft-rot fungi (subdivided into type I and II) as well as litter-decomposing and dung-dwelling (coprophilic) fungi; the latter two groups colonize the topsoil and the former ones, compact wood (trees, trunks, stumps) (Sutherland & Crawford, 1981; Nilsson et al., 1989; Blanchette, 1995; Dix & Webster, 1995; Hatakka, 2001; Hatakka & Hammel, 2010). Of course, there is some overlap of roles within these groups and certain fungal species (e.g. Hypholoma fasciculare) can colonize both habitats (Šnajdr et al., 2010). Furthermore, some species cause a ‘nonspecific’ rot that cannot be unequivocally assigned to a certain type; fungi dwelling in partially decayed wood often cause such nonspecific forms of rot (Dix & Webster, 1995).

To accomplish lignocellulose degradation, fungi actively secrete different sets of oxidative and hydrolytic biocatalysts along with low-molecular mass effectors (Hatakka & Hammel, 2010). Thus, white-rot basidiomycetes are known to secrete ligninolytic peroxidases, which belong to class II (secretory fungal peroxidases) of the superfamily of plant and microbial heme-peroxidases (also referred to as peroxidase-catalase superfamily; Zámocký & Obinger, 2010). Among them are the ligninolytic enzymes, manganese peroxidase (MnP, EC 1.11.1.13), lignin peroxidase (LiP, EC 1.11.1.14) and versatile peroxidase (VP, EC 1.11.1.16) (Hofrichter et al., 2010). Copper-containing phenol oxidases (laccase, EC 1.10.3.2) are another group of extracellular oxidoreductases which, in contrast to ligninolytic peroxidases, are frequently found in both ascomycetes and basidomycetes colonizing wood and litter. Laccases have diverse functions and are thought to be involved in polymerization–depolymerization processes of lignin, melanin and humic substances (Thurston, 1994; Leonowicz et al., 2001; Kluczek-Turpeinen et al., 2005; Baldrian, 2006; Liers et al., 2007; Hatakka & Hammel, 2010). Aryl alcohol oxidase (AAO, EC 1.1.3.7) is another extracellular oxidoreductase thought to be involved in ligninolysis and known to supply hydrogen peroxide (H2O2), the cosubstrate of ligninolytic peroxidases (Farmer et al., 1960; Guillén et al., 1992; Peláez et al., 1995).

LiP was the first ligninolytic enzyme described (for the corticoid basidiomycete Phanerochaete chrysosporium) and shown to have a redox potential that is high enough to directly oxidize the recalcitrant nonphenolic lignin subunits (e.g. arylglycerol-β-aryl-ether bonds) representing the most abundant structure in natural lignin (Glenn et al., 1983; Tien & Kirk, 1983). These in vitro studies were performed with lignin model compounds (e.g. adlerol) and synthetic lignins (Gold et al., 1984; Huynh et al., 1986; Hammel et al., 1993; Lundell et al., 1993).

The second ligninolytic enzyme, MnP, was first reported for P. chrysosporium and shown to preferentially oxidize phenolic lignin moieties (Glenn & Gold, 1985; Paszczyński et al., 1985; Tuor et al., 1992). Later, more than 50 basidiomycetous wood-rotters and litter-decomposers, including several families of agarics (Hatakka, 2001; Hofrichter, 2002), were shown to produce MnP. In contrast, the production of LiP appears to be restricted to a few corticoid and polyporous fungi (Hofrichter et al., 2010). MnP primarily oxidizes manganese(II) ions (Mn2+) into reactive Mn3+ that is chelated with organic acids and in turn oxidizes lignin and many other compounds (Hofrichter, 2002). It was shown that the enzyme is capable of depolymerizing and even mineralizing synthetic and natural polymeric lignins, lignin model compounds as well as humic substances (Forrester et al., 1988; Hofrichter et al., 1999, 2001; Kapich et al., 1999, 2005; Kästner & Hofrichter, 2001).

VP, the third ligninolytic peroxidase, is typically found in the genera Pleurotus and Bjerkandera and combines strong Mn2+-oxidizing activity with moderate activities towards phenolic and nonphenolic substrates and hence represents a functional hybrid of MnP and LiP (Heinfling et al., 1998; Mester & Field, 1998; Ruiz-Dueñas & Martínez, 2010).

LiP and Mn2+-independent VP activity, typically measured by the oxidation of veratryl alcohol (VA), is difficult to detect in lignocellulose extracts and thus there are only a few reports demonstrating their secretion in natural substrates (wood, straw) (Datta et al., 1991; Vares et al., 1995; Mester et al., 1998). In contrast, Mn2+–oxidation in wood, litter or soil extracts can easily be followed directly or in coupled assays (e.g. using Mn2+ and ABTS (2,2′-azino-bis(3-ethylthiazoline-6-sulfonate) or 3-methyl-2-benzo-thiazolinone hydrazone and 3,3-dimethylaminobenzoic acid), which allows to detect the activity of manganese-oxidizing peroxidases (MnOPs=MnP+VP) in natural substrates (Hofrichter, 2002; Steffen et al., 2007; Valášková et al., 2007). As MnOPs are mainly found in basidiomycetous fungi causing wood-decay or leaflitter decomposition, Mn2+–oxidation is a functional key activity (Lundell et al., 2010).

Over the last years, novel peroxidases secreted by saprobic basidiomycetes have been discovered, for example aromatic peroxygenases (APOs, EC 1.11.2.1; Ullrich et al., 2004; Hofrichter et al., 2010) and nonspecific manganese-independent peroxidases (MiP) that were later assigned as dye-decolorizing peroxidases (DyPs, EC 1.11.1.19; Kim et al., 1995; Sugano, 2009; Liers et al., 2010). APOs catalyze diverse oxygen transfer reactions which can result in the cleavage of ethers (Kinne et al., 2009; Hofrichter & Ullrich, 2010). DyPs oxidize recalcitrant nonphenolic substrates such as lignin model dimers and anthraquinone derivatives (Sugano, 2009; Liers et al., 2010). Although these reactions could be of relevance for the bioconversion of lignin, the actual physiological function of APOs and DyPs is still unclear (Liers et al., 2009; Hofrichter et al., 2010) and therefore studies are needed that follow their production in natural substrates (lignocelluloses) during fungal colonization. In this context, it is important to mention that more than 500 hits of APO- and DyP-like sequences can be found in genetic databases (although only a few APO/DyP proteins have been purified and characterized so far), indicating their widespread occurrence in the fungal kingdom (Pecyna et al., 2009; Hofrichter & Ullrich, 2010; Hofrichter et al., 2010; Ruiz-Dueñas & Martínez, 2010).

Many studies on the biodegradation of lignocelluloses by fungi have been based on the analysis of the structural wood components (lignin, cellulose, hemicelluloses) that remain after fungal colonization (Akhtar et al., 1997; Worrall et al., 1997; Jääskeläinen et al., 2003; Fukasawa et al., 2005; Verma et al., 2009). Standard methods such as the determination of Klason lignin or acid detergent lignin (ADL) use the gravimetric measurement of isolated and partially acid-hydrolyzed cell-wall material (Van Soest, 1963; Allen, 1989). To provide additional information on the effect of fungi and their enzymes on lignocelluloses, high-performance size-exclusion chromatography (HPSEC) has proved to be a reliable method to analyze the molecular mass distribution of water-soluble lignocellulose fragments released during wood decay or litter decomposition (Hofrichter et al., 2001; Steffen et al., 2002; Baumberger et al., 2007).

Here, we compare the patterns of released water-soluble lignocellulose fragments and secreted oxidative enzymes in the course of beech-wood colonization by 11 representative fungal species of different ecophysiological and taxonomic groups. Among them are typical white-rot basidiomycetes colonizing dead wood (Auricularia auricula-judae, Bjerkandera adusta, Phlebia radiata, Pleurotus eryngii, Pycnoporus cinnabarinus), the agaric litter-decomposer Stropharia rugosoannulata and nonspecific wood-rot fungi either colonizing hard wood (Agrocybe aegerita), inhabiting the old and already decayed wood (Mycena haematopus), or the residual lignocellulose in dung (Coprinellus radians). The soft-rot ascomycetes Xylaria polymorpha and Xylaria hypoxylon colonizing hard-wood were also included in the study.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Organisms and culture conditions

The fungal strains used are deposited in one of the following collections: the German collection of microorganisms and cell cultures (DSMZ, Braunschweig, Germany; A. aegerita 22459, A. auricula-judae 11326, C. radians 888, P. eryngii 9619), in the culture collections of the Department of Applied Chemistry and Microbiology (University of Helsinki, Finland; M. haematopus Hin 5, P. radiata K 233) and the Department of Bio- and Environmental Sciences (International Graduate School of Zittau, Germany; B. adusta Zi47, S. rugosoannulata Zi297, X. hypoxylon Zi002, X. polymorpha Zi005) or in the American Type Culture Collection (Manassas; P. cinnabarinus 200478).

The fungal organisms were routinely precultured on 2% malt-extract agar supplemented with beech-wood meal for 4 weeks, and three agar plugs (8 mm in diameter) of the overgrown plates were used to inoculate solid-state microcosms. These contained 3 g of sterilized beech-wood shavings (particle size <1 mm) and 10 mL of distilled water in 100-mL Erlenmeyer flasks (sterilization: 2 × 30 min at 120 °C and 2 bar; Liers et al., 2006). The flasks were incubated at 23 °C for about 10 weeks. After 4, 8, 12, 18, 24, 36 and 72 days, three flasks each were harvested and extracted with 20 mL of distilled water by vigorous shaking on a rotary shaker at 150 rpm for 4 h. Aqueous extracts were centrifuged and used for the measurement of extracellular enzyme activities and for the determination of the molecular mass distribution of soluble aromatic lignocellulose fragments formed during fungal growth.

Enzymatic activities

Activities of oxidoreductases were photometrically measured in 1-mL cuvettes in the presence of Mn2+ (0.5 mM MnCl2; MnOPs) or their absence (MiP activity), as well as with H2O2 (0.1 mM) for peroxidases and, respectively, without H2O2 for oxidases (laccase and AAO) (Wariishi et al., 1992; Eggert et al., 1996; Hofrichter et al., 1998; Liers et al., 2010), and were later corrected by each other. For example, the activities of laccase and MiP (later recognized as DyP-type peroxidase) were determined by following the oxidation of ABTS (ɛ420=36 mM-1 cm-1) in a sequential assay in 50 mM sodium malonate buffer at pH 4.5, and the peroxidase activities were corrected by those of laccase (Liers et al., 2010). DyP activity was confirmed by the specific Reactive Blue 5 (RBlue5) assay according to Shimokawa et al. (2008). MnP activity was specifically assayed as described previously by monitoring the formation of Mn3+–malonate complexes at 270 nm (Wariishi et al., 1992). Activities of LiP, APO and AAO were detected by following the oxidation of VA into veratraldehyde (ɛ310=9.3 mM-1 cm-1) at the corresponding pH optima [pH 3.0 for LiP (Kirk et al., 1986) as well as pH 7.0 for APO (Ullrich et al., 2004) and AAO (Muheim et al., 1990)] with and without H2O2 (1 mM), respectively. In all cases, the mean values of triplicate determinations were calculated and expressed in international units (U) defined as the amount of enzyme that forms 1 μmol product min−1 under the assay conditions used; enzyme activities are given per gram of dry mass of the particular beech-wood sample. All chemicals used were purchased from Sigma-Aldrich (Steinheim, Germany) and Merck (Darmstadt, Germany).

Analysis of water-soluble wood fragments

HPSEC was used to determine the molecular mass distribution of aromatic lignocellulose fragments formed as a consequence of fungal treatment (Hofrichter et al., 2001; Liers et al., 2006). The HPLC system (HP 1090 Liquid Chromatography; Hewlett-Packard, Waldbronn, Germany) was equipped with a diode array detector (HP 1100) and fitted with an HEMA-Bio linear column (8 × 300 mm, 10 μm) from Polymer Standard Service (Mainz, Germany). The mobile phase consisted of 20% acetonitrile and 80% of an aqueous solution of 0.34% NaCl and 0.2% K2HPO4; the pH was adjusted to 10 by adding NaOH (Hofrichter et al., 2001; Liers et al., 2006). The following separation parameters were used: flow rate 1 mL min−1, detection wavelength 280 nm and injection volume 20 μL; sodium polystyrene sulfonates (0.8–150 kDa, Polymer Standard Service), lignosulfonate (Biotech Lignosulfonate GesmbH, St Valentin, Austria) and alkaline lignin (Sigma-Aldrich) were used as molecular mass and UV-Vis standards and references, respectively.

Changes in the composition of the residual beech-wood were analyzed after 72 days of fungal cultivation. Three flasks per fungus were harvested as described above and the residual solids were used for lignocellulose analysis. Wood samples were dried at 60 °C to constant mass, weighted and ground by a planetary ball mill (5 min, 650 rpm, Pulverisette 7, Fritsch, Idar-Oberstein, Germany). Before lignin determination, samples were pretreated with acetone by accelerated solvent extraction (ASE Dionex 200, Idstein, Germany) at 100 °C and 1500 psi to remove wood extractives (Thurbide & Hughes, 2000). Klason lignin content was measured gravimetrically as the dry mass of solids after sequential hydrolysis with sulfuric acid (first step 72%; second step 4% H2SO4; Effland, 1977). For cellulose and hemicellulose analysis, degraded wood samples were analyzed with a FibertecTM analyser (FOSS Group, Rellingen, Germany) according to the modified Van Soest method (Van Soest, 1963; Naumann & Bassler, 1976) by the Landwirtschaftliche Kommunikations- und Servicegesellschaft mbH (Lichtenwalde, Germany). Resulting neutral detergent fiber (NDF) and acid detergent fiber (ADF) fractions as well as ADL were used finally for the calculation of cellulose (ADF−ADL) and hemicellulose (NDF−ADF) amounts. The loss of dry mass in fungal cultures was calculated as the mass difference compared with controls of beech-wood (initial Klason lignin, cellulose and hemicellulose content 19%, 31% and 48%, respectively) that were not treated with fungi. The fungal selectivity for the degradation of the wood components is given as lignin loss per total weight loss ratio (L/W) and hemicellulose per cellulose loss ratio (H/C).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Activities of fungal oxidoreductases during beech-wood colonization

Beech-wood shavings provided good support for growth of all studied basidio- and ascomyceteous fungi, which became visible by the development of fungal mycelia covering the whole woody material within 5–14 days. Wood degradation caused by white-rot fungi (e.g. B. adusta, A. auricula-judae, P. cinnabarinus and P. radiata) had a strong brightening effect on wood that started 1 week after inoculation. All basidiomycetes causing such a strong white-rot secreted MnOPs (measured directly by the formation of Mn3+–malonate complexes) with titers from 70 mU g−1 (S. rugosoannulata) up to 2045 mU g−1 (B. adusta; Table 1). Enzyme secretion was accompanied by a characteristic decrease in pH from approximately pH 5 to pH 3.5 (Fig. 1a), which was observed for all MnOP-producing fungi. Interestingly, the polyporous white-rot fungus P. cinnabarinus, which is known to predominantly produce laccase, also secreted MnOP (maximum level 125 mU g−1 reached on day 8 of cultivation) and caused the characteristic shift of pH below 4 (Fig. 1b). The jelly fungus A. auricula-judae, an underinvestigated white-rot fungus, secreted besides high MnOP titers (1750 mU g−1), an MiP (335 mU g−1) oxidizing ABTS in the absence of Mn2+ (Fig. 1c). As the latter activity also oxidized RBlue5 (maximal activity 41 mU g−1 on day 24), we concluded that the MiP activity must be a DyP-type peroxidase.

Table 1.  Maximum activities of oxidoreductases secreted by different lignocellulose-degrading basidiomycetes and ascomycetes during growth on beech-wood
FungusMaximum activity (mU g−1)
LaccaseAAOLiPMnOPMiPAPO
  1. Values given are means of three replicates (SD<5%).

Basidiomycetes
 Phlebia radiata26634 494  
 Pycnoporus cinnabarinus22  125  
 Bjerkandera adusta 218 2045  
 Pleurotus eryngii28913 362  
 Auricularia auricula-judae20  1758335 
 Mycena haematopus   72  
 Stropharia rugosoannulata   71  
 Agrocybe aegerita232    125
 Coprinellus radians14    20
Ascomycetes
 Xylaria polymorpha13     
 Xylaria hypoxylon6     
image

Figure 1.  Time course of the production of extracellular oxidoreductases by lignocellulose-degrading ascomycetes and basidiomycetes during growth on beech-wood: Phlebia radiata (a), Pycnoporus cinnabarinus (b), Auricularia auricula-judae (c), Agrocybe aegerita (d), Coprinellus radians (e), Xylaria polymorpha (f). Oxidase activities: black lines, laccase (triangles), AAO (diamonds). Peroxidase activities: red lines, MnOP (circles), MiP (diamonds), APO (triangles). Black dotted line=pH value.

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The nonspecific wood-rotters A. aegerita and C. radians produced moderate amounts of APO (20–125 mU g−1; Table 1) in the beech-wood microcosms, activity starting after, respectively, 24 days (A. aegerita, Fig. 1d) and 12 days (C. radians, Fig. 1e). During the secretion of APO, the pH of the cultures dropped slightly (e.g. from 5.3 to 5.1 in the case of A. aegerita).

Most basidiomycetes tested produced laccase with maximum activities varying between 14 mU g−1 (C. radians) and 289 mU g−1 (P. eryngii; Table 1). In all cases, maximum laccase levels were observed in the early stages of cultivation, on day 4 in the case of P. radiata and on day 8 in the case of A. aegerita, P. cinnabarinus, A. auricula-judae and C. radians (Fig. 1). No laccase activity was detected for the nonspecific wood-rotter M. haematopus and the litter-decomposer S. rugosoannulata.

Peroxide-producing AAO was only detected for the white-rot fungi P. radiata (34 mU g−1), B. adusta (218 mU g−1) and P. eryngii (13 mU g−1). A peroxide-dependent activity that oxidizes VA at pH 3.0, which would indicate the presence of LiP, was not detectable either in the wood extracts of strong or in moderate wood-rotters.

Laccase was the only oxidoreductase secreted by the soft-rot fungi X. hypoxylon and X. polymorpha with low activity maxima of 13 and 6 mU g−1, respectively (Table 1; Fig. 1f). The pH decreased slightly (e.g. from 5.1 to 4.9 in case of X. polymorpha) indicating that these ascomycetous fungi produced less organic acid than the basidiomycetes did. Whereas the wood-bleaching effect caused by Xylaria spp. was negligible, both fungi formed characteristic melanin lamellae, which eventually drew through the whole wood microcosm.

Release of water-soluble material loss of mass during fungal growth on beech-wood

HPSEC elution profiles of aqueous wood extracts revealed that, in comparison with an uninoculated control, the amount of water-soluble material was considerably increased as the result of fungal activity. The enzymatic attack resulted in the partial breakdown of the cell-wall network and the depolymerization of the lignocellulose constituents into smaller fragments. The aromatic nature of these water-soluble degradation products was proved by their spectral characteristics, which were compared with those of authentic lignin standards (UV-spectra are given in Fig. 2c).

image

Figure 2.  Selected HPSEC elution profiles of water-soluble aromatic lignocellulose fragments released after 72 days of fungal growth on beech-wood by peroxidase/peroxygenase producers (a) and laccase producers (b), black dotted line = control (beech-wood without fungus). UV spectra of water-soluble, aromatic lignocellulose fragments (black line) and of suitable reference substances [alkaline- (1, 4) and acid-soluble (2, 3) lignin, red line] (c).

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The predominant molecular mass of the water-soluble aromatics released by all white-rot basidiomycetes (except C. radians, which showed low overall oxidative enzyme activity; <25 mU g−1) was around 0.8 kDa and increased during the course of cultivation (Fig. 2a). Two representative HPSEC elution profiles recorded after 12 and 72 days are given in Fig. 3a–c and illustrate the molecular mass distribution of water-soluble fragments released by the MnOP-producing white-rot fungi P. cinnabarinus and A. auricula-judae, and the APO-producing nonspecific wood-rotter A. aegerita. The appearance of the characteristic low-molecular mass lignocellulose fragments (0.5–1 kDa) was observed, at least in the final stage (after 72 days), for all basidiomycetes except C. radians.

image

Figure 3.  Comparison of the patterns of water-soluble, aromatic lignocellulose fragments after different treatment times of fungal beech-wood with Pycnoporus cinnabarinus (a), Auricularia auricula-judae (b), Agrocybe aegerita (c), Xylaria polymorpha (d). HPSEC elution profiles were recorded after 12 days (thin line) and 72 days (thick line); black dotted line = control (beech-wood without fungus).

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Considerably more soluble high-molecular mass material was detected in the wood extracts of the ascomycetes that only produce laccase (Xylaria spp.) and also in extracts of the dung-isolate C. radians (Fig. 2b). Time-dependent changes in the molecular mass distribution caused by the soft-rot ascomycetes started on day 12, and a medium- as well as a high-molecular mass fraction (approximately 3 and 30 kDa) could be distinguished; the amount of both fractions increased permanently until the end of the experiment (Fig. 3d). After 10 weeks, both Xylaria spp. had additionally released a small fraction with very high-molecular mass (∼200 kDa), which may be an indication of polymerization processes associated with the formation of melanin layers in late cultivation stages.

It is an interesting fact that laccase-secreting basidiomycetes (A. auricula-judae, A. aegerita, C. radians, P. cinnabarinus, P. eryngii, P. radiata) also formed high-molecular mass fragments (30–32 kDa) in early culture stages (after 4–12 days). In the further course of fungal growth, the corresponding HPSEC peaks disappeared again and the characteristic low-molecular mass peak (∼0.8 kDa) of white-rot fungi appeared (e.g. Fig. 3b and c). As distinguished from these laccase producers, interim high-molecular mass peaks were not observed for B. adusta and they were very small for S. rugosoannulata and M. haematopus (data not shown). It is noteworthy that these three fungi did not produce laccase under the present culture conditions.

The highest total loss of mass in terms of total wood (0.5–0.16 g g−1) and lignin (0.72–0.16 g g−1) was observed for the white-rot fungi secreting activities of MnOP (P. radiata, P. cinnabarinus, B. adusta, A. auricula-judae) as well as for the nonspecific wood-rotter M. haematopus and the litter-decomposer S. rugosoannulata (Table 2). Although P. eryngii also secreted moderate levels of a MnOP, only small total wood and lignin losses (0.09 and 0.10 g g−1, respectively) were detected. A few changes in wood composition were also observed for the nonspecific wood-rotters and peroxygenase-producers A. aegerita and C. radians, causing losses of up to 0.08 and 0.09 g g−1 in total weight and 0.05 and 0.02 g g−1 in lignin during the trial period of 72 days.

Table 2.  Total mass loss and the loss of Klason lignin, hemicelluloses and cellulose after 72-day degradation of beech-wood by saprobic fungi
FungusWeight loss* (g g−1 of the original weight)L/WH/C
Total lossLigninHemicelluloseCellulose
  • The data represent average values (n=3), SD is given in parentheses.

  • *

    Total loss of structural wood components in relation to a control that represents the initial content of lignin, hemicelluloses and cellulose.

  • Xylaria spp. produced melanin which was detected together with Klason lignin and therefore affected the lignin content and masked the actual lignin loss.

Phlebia radiata0.41 (± <0.01)0.68 (± 0.01)0.61 (± 0.03)0.16 (± 0.01)1.73.8
Pycnoporus cinnabarinus0.50 (± 0.01)0.50 (± <0.01)0.64 (± 0.01)0.42 (± 0.03)1.01.5
Bjerkandera adusta0.31 (± 0.01)0.44 (± 0.03)0.37 (± 0.03)0.21 (± 0.01)1.41.8
Pleurotus eryngii0.09 (± 0.05)0.10 (± 0.01)0.09 (± 0.06)0.09 (± 0.07)1.11.0
Auricularia auricula-judae0.16 (± 0.01)0.16 (± 0.01)0.19 (± 0.01)0.13 (± 0.01)1.01.5
Mycena haematopus0.37 (± 0.02)0.72 (± 0.01)0.53 (± 0.04)0.14 (± 0.03)1.93.8
Stropharia rugosoannulata0.19 (± 0.01)0.22 (± 0.05)0.24 (± 0.03)0.14 (± 0.02)1.21.7
Agrocybe aegerita0.08 (± 0.02)0.05 (± 0.04)0.12 (± 0.05)0.06 (± 0.02)0.62.0
Coprinellus radians0.09 (± 0.01)0.02 (± 0.02)0.09 (± 0.01)0.12 (± 0.03)0.20.8
Xylaria polymorpha0.09 (± 0.02)0.16 (± 0.02)0.09 (± 0.03)1.8
Xylaria hypoxylon0.16 (± 0.03)0.18 (± 0.04)0.23 (± 0.07)0.8

The soft-rot fungi tested also caused moderate losses in total wood mass (e.g. 0.16 g g−1 for X. hypoxylon), which is in the same range as the loss of mass caused by A. auricula-judae. An effect on the Klason lignin content was not detectable for any Xylaria spp.; probably it was masked by the concomitant formation of melanin.

The loss in mass in hemicelluloses caused by the white-rot fungi (0.64–0.19 g g−1) was higher than that observed for cellulose (0.42–0.16 g g−1), reflecting the characteristic features of white-rot decay, during which the whitish cellulose fibers remain. The two soft-rot fungi and the nonspecific wood-rotters A. aegerita and C. radians utilized both hemicelluloses (0.09 and 0.18 g g−1) and cellulose (0.06 and 0.23 g g−1).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Nine of the 11 tested fungal species produced extracellular peroxidases during growth in beech-wood microcosms. Among them, seven species secreted MnOPs, two species APOs and one species an MiP of the DyP-type. MnOP activities detected varied between 71 mU g−1 (S. rugosoannulata) and 2045 mU g−1 (B. adusta), which is in the range of previously reported data for white-rot fungi grown on natural lignocelluloses such as wheat straw, spruce, pine and aspen wood. For example, 200–3000 mU g−1 were reported for Ganoderma applanatum (Dinis et al., 2009) and up to 7000 mU g−1 for Ceriporiopsis subvermispora (Messner et al., 1998). The secretion of MnOP was accompanied by a characteristic acidification of the microcosms, which is typical for this type of wood-rot and is caused by organic acids (e.g. oxalate, malate) secreted by the fungi to chelate Mn3+-ions (Hofrichter et al., 1998).

The lack of VA-oxidizing activity at pH 3 indicates that either no LiP was secreted (as known to be produced by P. eryngii, P. radiata and B. adusta) or that the activity was masked by wood ingredients (Vares et al., 1995; Camarero et al., 1999); the latter may also be valid for the VA-oxidizing activity of VP, which is about two to three orders of magnitude lower than the Mn2+-oxidizing activity (Martínez et al., 1996; Mester & Field, 1998; Ruiz-Dueñas et al., 2001). Thus, our findings strongly support the assumption that peroxidases oxidizing Mn2+-ions are the most abundant ligninolytic peroxidases secreted by wood-rot basidiomycetes (Hatakka, 1994; Hofrichter, 2002).

Detection of the recently described peroxidative activities of A. auricula-judae (DyP-type peroxidase activity approved by RBlue5 oxidation; Kim et al., 1995; Liers et al., 2010) and A. aegerita as well as C. radians (APO activity measured by the oxidation of VA at pH 7.0) in beech-wood cultures, strengthens the assumption that fungal DyPs and APOs may be involved somehow in the conversion of lignin and/or its degradation products (Liers et al., 2010; Kinne et al., 2011).

The production of extracellular peroxidases by wood-rot fungi has been often followed in synthetic and defined liquid media (Niku-Paavola et al., 1990; Hatakka, 1994; Eggert et al., 1996; Steffen et al., 2002), but the patterns of enzyme secretion in solid woody substrates better reflect natural conditions and may allow the identification of the key enzymes involved in lignin degradation. For example, the well-studied white-rot fungus P. cinnabarinus was found to secrete only laccase when growing in synthetic liquid media (Eggert et al., 1996) but in the beech-wood cultures studied here, it also secreted substantial levels of an MnOP activity. The fungus had been supposed to decompose wood exclusively by the action of high-redox potential laccases in cooperation with the fungal metabolite hydroxyanthranilic acid acting as low-molecular mass redox mediator (Eggert et al., 1996, 1997; Herpoël et al., 2000). Our finding of MnOP activities in wood extracts of P. cinnabarinus and the identification of MnP genes in the fungus (Dölz et al., 2007; Morgenstern et al., 2010; Uzan et al., 2010) strongly suggest that it belongs to the abundant group of MnP-laccase-producing white-rot fungi (Hatakka, 1994, 2001; Eggert et al., 1997; Hatakka & Hammel, 2010).

AAO activities could be detected in beech-wood cultures of three white-rot fungi including P. radiata. AAOs from basidiomycetes are only known for P. eryngii (Guillén et al., 1992, 1994), B. adusta (Muheim et al., 1990) and Trametes versicolor (Farmer et al., 1960), whereas a respective enzyme of P. radiata has not been detected so far. Furthermore, this is to our best knowledge the first evidence of AAO production in wood; up to now, the secretion of Pleurotus AAO has only been demonstrated in a wheat-straw culture (Camarero et al., 1997).

In the present study, APO was detected for the first time under conditions close to nature, i.e. in slightly acidic beech-wood microcosms with a pH 5.3 for A. aegerita and 5.8 for C. radians. So far, the production of APO has only been reported for C- and N-rich complex media such as soybean suspensions where the pH tends to increase above 8 during cultivation (Ullrich et al., 2004; Dau et al., 2007; Hofrichter et al., 2010). It should be noted that APO is able to cleave ethers including nonphenolic lignin model compounds (Kinne et al., 2009, 2011), and that the peroxygenase model fungus, A. aegerita, can mineralize and solubilize 14C-labeled lignin to some extent (Liers et al., 2009, unpublished data). The activity of both nonspecific wood rotters on beech-wood was further demonstrated by the total mass loss (7% in both cases) and lignin loss (2–4%) as well as by the conversion of wood polysaccharides, in the range of data determined for the less efficient white-rotter P. eryngii (e.g. 8–10% for hemicellulose and 6–10% for cellulose).

The differences in the fragmentation patterns of the HPSEC elution profile accordingly indicate the oxidative effect of peroxidases on milled lignocelluloses and isolated lignins (formation of ∼0.8 kDa fragments), in contrast to the mainly polymerizing activities of laccases (Rittstieg et al., 2002; Guebitz & Cavaco-Paulo, 2008; Mattinen et al., 2008) (Fig. 3). The interim release of water-soluble fragments with a mass around 30 kDa was observed for all laccase-producing basidiomycetes, but only in the initial culture stage. In contrast, the soft-rot fungi of the genus Xylaria preferentially produced such fragments and even larger ones (up to 200 kD) along with medium-sized fragments (∼3 kDa) throughout the cultivation, which may be explained by the secretion of laccase as the only lignin-modifying enzyme. Thus, laccase may be involved in the oxidation of phenolic structures, leading to the polymerization of lignin and melanins (Li et al., 1999; Schwarze et al., 2000). The latter was deduced from the formation of melanin lamellae in the cultures of both fungi and corresponds to previous observations reported for Xylaria spp. (Liers et al., 2006, 2007), Paecilomyces inflatus (Kluczek-Turpeinen et al., 2007) and Daldinia concentrica (Shary et al., 2007).

The pronounced activities of MnOPs may enable the white-rotters to initiate efficient lignin removal in the beech-wood, which is substantiated by a high L/W ratio (between 1.0 and 1.9). These values fit to the few data reported for other basidiomycetous fungi (e.g. Mycena spp., Daedalea spp., T. versicolor and Trametes hirsuta reached L/W ratios between 1.0 and 1.7; Fukasawa et al., 2005). Relatively large weight losses in lignin (measured as acid-soluble Klason lignin) have also been reported for other white-rot fungi grown in solid-state cultures on different lignocelluloses, for example 12% for P. eryngii and 55% for P. chrysosporium (Martínez et al., 1994), 16% for Auricularia spp. (Hakala et al., 2004), 60% and 27% for Lentinula edodes and Mycena spp. (Osono & Takeda, 2002), respectively, and 37% for Hypholoma spp. (Valášková et al., 2007).

The present study demonstrates that white-rot fungi produce MnOPs as the predominant enzymes of ligninolysis. Whereas no LiP was detected in the wood extracts, activities of two recently discovered peroxidases, APO (A. aegerita, C. radians) and DyP-type peroxidase (A. auricula-judae), were demonstrated for the first time. In general, solid-state cultivation of fungal organisms on woody materials in microcosms is a suitable method for the identification of enzymatic key activities contributing to ligninolysis which may otherwise be overlooked, for example, the oxidation of manganese by P. cinnabarinus. The activity of peroxidases seemingly correlated with lignin removal (2–13% determined by the Klason method) and the release of small (0.5–1 kDa), water-soluble lignocellulose fragments of an aromatic nature (absorption band at 280 nm) as demonstrated by HPSEC. These patterns differ from the characteristic high-molecular mass fragments (30–200 kDa) released by ascomycetes (e.g. X. polymorpha), which only secreted laccase under the conditions used.

In summary, evaluation of oxidative biocatalyst production combined with analysis of changes in the chemical composition of wood and the patterns of released fragments allows differentiation between the different ecophysiological groups of lignocelluloses degrading fungi and may be useful to identify fungal key species.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The work was financially supported by the Deutsche Forschungsgemeinschaft (DFG Priority Program 1374 – ‘Infrastructure & Biodiversity Exploratories’); Fupers (HO1961/4-1) and Funwood (HO1961/5-1), the European Union (integrated projects Biorenew, Peroxicats) and the Deutsche Bundesstiftung Umwelt (DBU, Project 13211-32 ‘Pilzliche Sekretome’). We thank Torsten (Ed) Adam and all coworkers for useful comments and their know-how.

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  6. Discussion
  7. Acknowledgements
  8. References
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