Interactions of heavy metals with white-rot fungi

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Abstract. White-rot fungi require trace amounts of essential heavy metals such as Cd, Mn or Zn for their growth, but these metals are toxic when present in excess ...
Enzyme and Microbial Technology 32 (2003) 78–91

Interactions of heavy metals with white-rot fungi Petr Baldrian∗ Laboratory of Biochemistry of the Wood-rotting Fungi, Institute of Microbiology AS CR, V´ıdeˇnská 1083, Prague 4 CZ-14220, Czech Republic Received 23 May 2002; received in revised form 19 September 2002; accepted 23 September 2002

Abstract White-rot fungi require trace amounts of essential heavy metals such as Cd, Mn or Zn for their growth, but these metals are toxic when present in excess. Toxic heavy metals can inhibit the growth, cause morphological and physiological changes and affect the reproduction of Basidiomycetes. Fungal species and strains differ in their sensitivity towards metals and in the protection mechanisms involved. The toxicity of some heavy metals such as Hg, Cu or Ni has been used for the development of antifungal wood preservatives. Extracellular ligninolytic and cellulolytic enzymes are regulated by heavy metals on the level of transcription as well as during their action. During the degradation of lignocellulose and xenobiotics by white-rot fungi or isolated enzymes from these fungi heavy metals interfere with both the activity of extracellular enzymes involved in the process and fungal colonization. The ability of white-rot fungi to adsorb and accumulate metals together with the excellent mechanical properties of fungal mycelial pellets provide an opportunity for application of fungal mycelia in selective sorption of individual heavy metal ions from polluted water. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Basidiomycetes; Cadmium; Copper; Bioremediation; Heavy metals; Laccase; Mercury; Mn-dependent peroxidase; Phanerochaete chrysosporium; Pleurotus ostreatus; Trametes versicolor; Sorption; White-rot fungi; Wood

1. Introduction White-rot fungi are characterized by their unique ability to degrade lignin. In addition to lignin degradation, white-rot fungi are also able to degrade a variety of structurally similar organic compounds. Various aspects of physiology, ecology and the biotechnological uses of these fungi have been studied. However, until now, there is only scattered information about the physiological effects of heavy metals on white-rot fungi. Only in recent years have the first studies concerning heavy metals effects on biotechnological processes performed by white-rot fungi appeared in literature. Some heavy metals are essential for the fungal metabolism, whereas others have no known biological role. Both essential and nonessential heavy metals are toxic for fungi, when present in excess. Whereas fungi have metabolic requirements for trace metals, the same metals are often toxic at concentrations only a few times greater than those required [1]. The metals necessary for fungal growth include copper, iron, manganese, molybdenum, zinc, and nickel. Nonessential metals commonly encountered include chromium, cadmium, lead, mercury and silver [2]. The involvement of metal ions in the physiology of another group ∗

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of wood-rotting fungi, the brown-rot fungi has currently been reviewed [3]. Metal ions are involved in the decomposition of cellulose and hemicelluloses by brown-rot fungi. In white-rot fungi, copper and manganese directly participate in the process of lignin degradation. Manganese participates in the reaction cycle of Mn-dependent peroxidase, and copper serves as a cofactor in the catalytic center of laccase. The role of Mn in lignin degradation has been the subject of numerous studies. In this review, attention will be paid mainly to the toxic heavy metals, that include all the nonessential metals and copper. In contrast with other essential metals, copper is toxic to most fungi even at very low concentrations. The fungi must be able to sequester essential trace metal ions from various sources, where the metals can be present in concentrations ranging from trace to toxic levels. The concentration of heavy metal ions in their main source, wood, is usually low. In beech (Fagus sylvatica), Cd and Pb concentrations are usually below 1 ppm, Zn concentration can reach tens of parts per million. This is 10–100 times less than in soils at corresponding sites [4]. White-rot fungi have to cope with toxic levels of metal ions often during their growth in soil. The concentration and availability of heavy metal ions in soil are generally higher than in wood, and the concentration can be greatly elevated as a result of industrial pollution on specific sites. Near motorways, gaswork sites, incineration plants and other industrial facilities,

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soil contamination with heavy metal ions is often accompanied by the presence of high levels of polycyclic aromatic hydrocarbons (PAHs) [5]. The in situ degradation of organic pollutants in such places by white-rot fungi poses a specific problem [6], because the fungal growth can be inhibited by some heavy metal ions. Fungal decomposition of wood treated with different metal-based preservatives is also problematic. Last, but not least, the fruit bodies of white-rot fungi receive significant amounts of heavy metals from the atmosphere.

2. Uptake of heavy metals Heavy metals present in the environment can directly interact with extracellular enzymes of fungi. However, to cause a physiological response, heavy metals must be taken up by the fungus. The uptake from liquid environment is the most simple situation, not only under laboratory conditions, but also in the case of water-containing substrates. White-rot fungi can concentrate metals taken up from substrate in their mycelia. Pleurotus ostreatus was able to accumulate 20 mg g−1 dry weight Cd from liquid medium containing 150 ppm Cd with at least 20% of accumulated Cd deposited intracellularly [7]. During 7-day growth in potato sucrose medium containing 5 mM of Cu and Zn, the fungus accumulated 10 ␮g g−1 dry weight Cu (2.4 ␮g g−1 protein in cytosol) and 5 ␮g g−1 dry weight Zn. In a 3 mM Cd medium, the fungus accumulated 20 ␮g g−1 dry weight Cd (2.8 ␮g g−1 protein in cytosol) [8]. During the growth of Daedalea quercina in media containing equimolar metals, the concentrations of the accumulated metal ions decreased in the order Zn > Cu > Pb > Al [9]. The preference for individual heavy metals is species-specific. During 8-day cultivation of four white-rot fungi D. quercina, Ganoderma applanatum, Stereum hirsutum and Schizophyllum commune in solutions containing Al, Cd, Pb, and Ca at 1 mM each, most Pb was found in S. hirsutum (90.6 ␮M g−1 ), most Cd, Al, and Ca in G. applanatum (272, 600, and 602 ␮M g−1 , respectively). From equimolar solution (1 mM), Pb was preferentially accumulated by all fungi except G. applanatum that accumulated more Al [10]. Although experiments with heavy metals accumulation from liquid media can provide useful data about the uptake capacity of fungi, they do not exactly reflect the situation in nature. In liquid cultures of Volvariella volvacea, Cu was taken up preferentially, Hg and Pb uptake was low. On the other hand, when grown on wheat substrate, the highest uptake by sporocarps was recorded in the case of Pb and the lowest with Cd [11]. The accumulation of metals by edible fungi can be a limitation for their use as food. About 3.5% Cd and 12% Hg was translocated into fruit bodies of Pleurotus cornucopiae from metal-supplemented straw substrate [12]. The fungus was able to concentrate Hg from artificially enriched substrate by a factor of 65–140. However, at high Hg concentrations (0.1–0.2 ppm), the growth was strongly

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reduced [13]. Sporocarps of another edible fungus Pleurotus sajor-caju accumulated high amount of Cd and little Pb [14]. Experiments using radioactive isotopes 109 Cd and 203 Hg revealed, that fungal fruit bodies communicate with their substrate. The communication between the substrate and fungal fruit bodies occurs in both directions. Metals applied into fruit bodies of Agrocybe aegerita were partially translocated into the substrate (wheat straw) and into consecutive harvests [15]. The amount of translocation of Cd and Hg by mycelium of A. aegerita decreased with increasing metal concentrations, probably due to toxicity [16]. White-rot fungi growing on wood in the nature were found to accumulate Cd, Fe, Zn and Cu from wood in their fruit bodies, whereas Mn and Pb were excluded. Similar results with metal accumulation/exclusion were also found with litter-decomposing and ectomycorrhizal soil fungi [17]. The accumulation of heavy metals also proceeds from soil, which is the natural substrate for many white-rot species. Sporophores of Armillaria mellea, collected from metal-polluted soils near motorways contained several parts per million of Cd and Pb and tens of parts per million of Zn. Cd was accumulated with a factor of 32, Zn and Pb were excluded, the concentration in sporophores reaching 30–40% of that in the topsoil [18]. The molecular mechanisms of heavy metals accumulation by white-rot fungi have not been studied. In other fungi, transporter systems for the uptake of essential metals are present in the cell membrane. The nonessential metals are usually cotransported using the same transporters due to their low specificity. In the ectomycorrhizal fungus Paxillus involutus, the uptake of Cd involves rapid binding to the cell wall and a slower, carrier-mediated transport into the cell. The uptake partially depends on the membrane potential and it is linked to the transport of Ca. On the subcellular level, 50% of metal are bound to the cell wall, 30% remain in the cytoplasm and 20% are transported into vacuoles [19]. The cotransport of nonessential bivalent metals with Ca can play a role also in white-rot fungi because the fruit bodies of most wood-rotting fungi contain high contents of Ca [20]. The presence of Cd/Cu cotransport is probable in P. ostreatus, since both Cu and Cu + glutathione significantly reduce Cd accumulation to toxic levels [7]. It has to be noted, that cadmium and copper exert their toxic effects directly on the plasma membrane, where they interfere with solute transport and other membrane phenomena. Both metals cause membrane permeabilization (associated with K efflux), as well as changes in the membrane composition [21].

3. Effects of heavy metals on the physiology of white-rot fungi 3.1. Growth and metabolic activity After heavy metals enter the fungal cell, they affect both individual reactions and complex metabolic processes.

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Growth is the most popular complex phenomenon, that was studied from the viewpoint of heavy metals toxicity. Cd and Hg are in general the most toxic metals for all white-rot fungi. In S. commune, addition of only 0.1–0.2 mM Cd led to severe growth inhibition [22], addition of 0.05–0.25 mM mercury to growing cultures of Phanerochaete chrysosporium led to decrease of growth rate and in higher Hg concentrations lysis of the mycelium occured, accompanied by the decrease in protein contents of the mycelia [23]. Mercury and cadmium exhibited also the highest toxicity towards S. hirsutum [24]. The essential metals are relatively less toxic. Growth of biomass of P. chrysosporium was limited in the presence of 50 ppm Ni, Cd, and Pb, whereas in the case of Co and Cu, decrease of the growth rate was apparent in 150 ppm and in the case of Mn in 300 ppm [25]. On the other hand, low concentrations of the essential metals Co, Cu and Mn increased slightly the growth rate of this species [26]. In Ganoderma lucidum, toxicity of heavy metals decreased in the order Hg > Cd > Cu > U > Pb > Mn = Zn [27]. The decrease of fungal growth rate is sometimes accompanied with the increase of the lag phase. Lengthening of lag phase was also recorded on media containing Hg in the case of Pycnoporus cinnabarinus [28]. Cadmium added to straw inoculated with Agrocybe perfecta in concentrations 0.01–1 mM decreased significantly the loss of organic matter, another complex phenomenon directly linked with the fungal growth. In Pleurotus ostreatoroseus, there was no effect of metal addition and in P. ostreatus, highest substrate decomposition was found at 0.5–1 mM Cd [29]. The presence of metals also interferes with the colonization of soil [30]. 3.2. Mycelial morphology The decrease of growth rate of fungi caused by heavy metals is often accompanied by morphological changes of the growing mycelium. The transfer of S. commune onto solid medium containing Cd led to an increase of aerial hyphae formation and to morphological changes in the growth segment of hyphae. Loops and connective filaments developed, together with an increase of hyphal branching. The extent of these changes reflected the increasing concentration of the metal [22]. Pellets of D. quercina, cultivated on liquid media with Cd exhibited shortening and irregular appearance of surface hyphae. The pellets in Cd-containing cultures also had smoother surface [9]. The morphological changes induced by heavy metals are common among all groups of fungi. In ectomycorrhizal fungus P. involutus, addition of Cd led to the increase in hyphal density caused by increased number of laterals per branch point and a decrease of the distance between branch points [31]. Fungal cultures growing in the presence of heavy metals can also change their discoloration. S. hirsutum produced a yellow-orange pigment both extracellularly and in the mycelium, when cultivated in the presence of 0.25 mM or more Cd [32], Trametes versicolor produced a brown pig-

ment on Cd-containing media [24]. During the cultivation of S. commune in liquid media containing Pb, black mycelial pellets formed, while the controls were creamy [10]. 3.3. Enzymatic activities Heavy metals in general are potent inhibitors of enzymatic reactions [33]. Mercury exerts its toxic effect mainly by binding to SH groups present in the active or regulation sites of enzyme and causing their irreversible inactivation. Copper and cadmium—in addition to binding to aromatic amino acid residues in enzyme molecules, can also cause oxidative damage of proteins by the induction of oxidative stress associated with the production of reactive oxygen species like hydroxyl or superoxide radicals [34]. In white-rot fungi, most attention has been paid to the metal toxicity towards extracellular enzymes. It is not surprising, since these involve the energy metabolism complex of cellulose and hemicellulose degrading enzymes as well as the ligninolytic enzymes, studied both from the viewpoint of fungal physiology and biotechnology. The enzymes produced into the extracellular environment often face high concentrations of metals, since they are not protected by the cell-associated metal-detoxication mechanisms. After entering the cell, metals can also influence the production of extracellular enzymes on the levels of transcriptional and translational regulation. It seems, that low concentrations of essential heavy metals are necessary for the development of the ligninolytic enzyme system. Addition of low concentrations of Zn (0.006–18 ␮M) and Cu (0.0004–1.2 ␮M) into the metal-free synthetic cultivation medium increased the activity of lignin peroxidase and Mn-peroxidase of P. chrysosporium. The metals also increased solubilization and mineralization of lignin [35]. Two heavy metals are directly involved in the reactions catalyzed by ligninolytic enzymes. The role of Mn in lignin degradation has been studied in detail. Manganese is directly involved in the catalytic cycle of Mn-dependent peroxidase (MnP) and it has been reported that Mn also plays a regulatory role in the expression of lignin peroxidase, MnP, laccase and in the degradation of lignin [36,37]. The other heavy metal, involved in lignin degradation is Cu, the cofactor of the enzyme laccase. Although the presence of copper in the catalytic center of the enzyme has been known for long, the important regulative role of copper in laccase production has only recently been addressed. The positive effect of copper addition on the production of laccase was observed in Ceriporiopsis subvermispora [38,39], T. versicolor [40], Marasmius quercophilus [41], P. ostreatus [42], P. chrysosporium [43], T. pubescens, T. multicolor, T. hirsuta, T. gibbosa, T. suaveolens, G. applanatum, Polyporus ciliatus, Panus tigrinus [44], P. sajor-caju [45], and T. trogii [46]. The enzyme is strongly regulated on the level of transcription [40,42,45]: the transcript level in T. versicolor increased within 15 min after Cu addition to Cu-free cultures [40]. The supplementation of P. ostreatus

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cultures with Cu increased the production of all isoenzymes of laccase produced by the fungus. However, the extracellular levels of the protein were low, probably due to the activity of specific proteases secreted into the copper-containing medium [42]. Metal responsive elements (MRE) were identified in the promoters of P. ostreatus laccase genes poxc and poxa1b. These MREs interact with Cu-responsive transcription factors. The organization of MRE sites is similar to that of metallothionein genes [47]. In P. chrysosporium and M. quercophilus, production of extra laccase isoenzymes, not present under natural conditions, was observed after Cu addition [43]. It has to be mentioned that P. chrysosporium produces low laccase levels and some strains do not produce this enzyme. The induction of laccase by other metals, that are able to cause oxidative stress was studied with Trametes pubescens. Only Mn and Cu enhanced laccase formation, whereas Ag, Cd, Hg and Zn were not effective in enhancing laccase formation [44]. In P. ostreatus laccase activity was also increased by the addition of 1–5 mM Cd. The rate of increase in laccase activity was dependent on the age of the culture. Addition of 1 mM Ag, Hg, Pb, Zn, and H2 O2 decreased activity of the enzyme. Copper also increased activity of the produced extracellular enzyme when added to the extract from straw culture. It is interesting to note that extracts from cultures of different age were different in sensitivity to copper addition. Furthermore, when added to purified laccase, Cu increased its stability in time. Addition of Hg to the purified enzyme led to an immediate loss of activity of the enzyme and in low concentrations it substantially decreased the temporal stability of the enzyme [48]. Recently, a novel extracellular protease of P. ostreatus PoS1 has been purified and showed to be responsible for laccase degradation. The activity of PoS1 is decreased to 77% in presence of 1 mM Cu [49]. This might explain the positive effect of Cu on enzyme stabilization in the extracts from straw cultures. Amongst others, fungal laccases are believed to be involved in the formation of various pigments [50,51]. Accordingly, one possible function of laccase could be the Cu-induced formation of melanin. When T. pubescens was grown in the presence of Cu, the mycelia turned dark brown [44]. Enhanced cell wall melanin accumulation as a response to elevated Cu concentrations was also described in ascomycetes [52]. Records about heavy metals effects on other ligninolytic enzymes are scarce. In T. trogii, addition of copper increased the activities of Mn-peroxidase and glyoxal oxidase, as well as the decolorization of the polymeric dye Poly R-478. Highest enzyme activities and decolorization rate was obtained with 1.6 mM Cu, the highest concentration tested [46]. The activity of all ligninolytic enzymes of S. hirsutum in liquid nitrogen-limited media decreased when cultivation proceeded in 0.25 mM Cd. The control exhibited laccase activity on days 3–21, whereas in Cd-treated samples, laccase was only present on days 6–12, and the peak activity on day 6 was lower. The activities of Mn-dependent peroxidase

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and Mn-independent peroxidases (MIP) were completely absent under these circumstances. Cadmium in concentrations of 0.5–1.0 mM also inhibited the activities of laccase, MnP and MIP of P. chrysosporium, whereas 0.1 mM Cd did not affect the activity of the enzymes. MnP was inhibited most. In P. chrysosporium, the activity of lignin peroxidase was inhibited in 1.0 mM Cd [32]. Cadmium is a reversible competitive inhibitor of Mn-dependent peroxidase. It also inhibits enzyme-generated Mn3+ -chelate-mediated oxidation of 2,6-dimethoxyphenol, but it does not inhibit direct oxidation of phenols. Cd protects MnP against thermal inactivation more efficiently than Ca, extending the half-life of the enzyme [53]. The general toxic effect of nonessential heavy metals also affects the activity of other extracellular enzymes, involved in ligninolysis. Aryl alcohol oxidase from P. eryngii is inhibited by Ag+ and Pb2+ [54]. Ag+ , Cu2+ , and Hg2+ inhibited and Fe2+ increased the activity of pyranose oxidase in an unidentified basidiomycete [55]. Ag+ and Hg2+ inhibited the same enzyme in P. chrysosporium [56]. In addition, Ag+ , Co2+ , Cu2+ , and Ni2+ also inhibited pyranose oxdiase in T. versicolor [57]. The presence of heavy metals can also interfere with the carbon and energy supplying system of cellulases and hemicellulases. Cellulase of P. chrysosporium in liquid media was inhibited in the presence of 50–150 ppm Cd, Cu, Pb, Mn, Ni, and Co. At 150–300 ppm Mn or 300 ppm Cd or Co, no cellulase activity was detected [25]. On the other hand, cadmium significantly increased the activity of endocellulase during the cultivation of P. ostreatus on straw [58]. Hg2+ , Fe3+ and Cu2+ strongly inhibited the activity of ␤-glucosidase from T. gibbosa [59]. Cu, Hg, Sn and Pb, but not Fe, Zn, Co or Mn also inhibited ␤-glucosidases isolated from the brown-rot fungi Gloeophyllum sepiarium and G. trabeum [60]. In ericoid mycorrhizal fungi Oidiodendron spp., inhibition of polygalacturonases by a wide range of metals was recorded [61]. These enzymes are involved in the degradation of middle lamellae of wood cell elements. Little is known about the effect of metals on intracellular enzymes of white-rot fungi. The activity of ribonuclease from P. tuber-regium was inhibited by Hg, Zn, Ni, Ca, and Pb [62]. On the other hand, superoxide dismutase belongs to the group of enzymes, that are usually induced in conditions causing oxidative stress, including the presence of heavy metals. In the ectomycorrhizal fungus P. involutus, Cd activated superoxide dismutase on the post-translational level. The enzyme is involved in cadmium stress response by decomposing the superoxide radical molecules [63]. 3.4. Reproduction In different taxonomic groups of fungi, it was found that heavy metals are harmful for reproduction. In saprophytic and mycorrhizal soil fungi, the reproductive stages of development (spore formation and germination) are much more sensitive to heavy metals than mycelial growth [64,65]. In white-rot fungi, the sensitivity of fruiting differs from

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species to species. A. perfecta failed to produce fruit bodies during growth on straw with 0.05–1 mM Cd, whereas P. ostreatus was much less sensitive [29]. Favero et al. [66] found sporocarps formation in another strain of the same species to be unaffected by up to 285 ppm Cd in the fungal substrate. Cd accumulated preferentially in caps (56 ppm compared to 36 ppm in stems). When straw was overlaid with nonsterile Cd-containing soil, A. perfecta and P. ostreatoroseus failed to produce fruit bodies at 50 ppm Cd, whereas P. ostreatus produced fruit bodies even at 500 ppm Cd. About 1–5% of the metal present in the soil was translocated into sporocarps [29]. Heavy metals, above all Cd, Hg and Co, were also found to be toxic for the sporocarp development of V. volvacea, the fungus cultivated for food [11]. Hg also caused a decrease of protein contents in sporocarps [14]. In a standard test of renewed fruiting from excised pilei, Cd and Mn reduced the yield of P. ostreatus fruit bodies. At 4.5 mM Cd, the yield of fruit bodies was reduced by 50%; about 20 ␮g g−1 Cd was accumulated [67]. 3.5. Defense mechanisms The interaction of fungi with heavy metals causes severe changes in the physiological processes and under certain circumstances it can even kill the mycelium. Therefore, fungi evolved active defense mechanisms, that alleviate the toxicity of metals. The defense is usually based on immobilization of heavy metals using extracellular and intracellular chelating compounds. In many different taxonomic groups of fungi, heavy metals are intracellularly chelated by peptidic low molecular weight compounds—phytochelatins or metallothioneins [68]. Although the production of these low molecular weight compounds was also detected in basidiomycetes [69,70], it seems, that their role in wood-rotting fungi is limited and their production by these fungi was never proven. In white-rot and brown-rot fungi, the extracellular metal chelation is probably more important. However, it is not clear, if this is an active defense process. One of the typical metal chelators produced by both white-rot and brown-rot fungi is oxalate. The production of oxalic acid by fungi provides a means of immobilizing soluble metal ions or complexes as insoluble oxalates, thus decreasing bioavailability and increasing tolerance to these metals [71]. Rabanus [72] and Shimazono and Takubo [73] suggested that metal tolerance of brown-rot fungi is linked to oxalic acid production, which presumably precipitates copper into the insoluble form of copper oxalate. A relationship between copper tolerance and oxalic acid production has been found to be due to copper oxalate crystal formation in decayed wood [74]. Copper oxalate crystals (moolooite) has been observed around hyphae of brown-rot fungi growing on wood treated with a copper-based preservative [74–76]. Metal oxalates can be also formed with Ca, Cd, Co, Cu, Mn, Sr, and Zn [71]. Although the production of oxalate is more typical for brown-rot fungi, it also plays an important role in the white-rot decay of wood with

lignin peroxidase [77], Mn-peroxidase of P. chrysosporium [78] or a low molecular weight compound contributing to the Fenton-based breakdown of wood components [79,80]. Among white-rot fungi, higher amounts of oxalate are produced by P. ostreatus, P. chrysosporium, and T. versicolor [80,81]. White-rot fungi produce extracellular hyphal sheath, composed mainly of polysaccharides. This mucilaginous extracellular matrix probably contributes to the facilitation of the joint action of different enzymes, involved in the degradation of lignocellulose substrate [82]. The material is made mostly of polysaccharides including ␤-1,3-glucan with ␤-1,6-linkages [83,84]. Calcium oxalate crystals have been found in this extracellular mucilaginous materials of different species including P. chrysosporium, T. versicolor, Phellinus noxious, or Rigidoporus lignosus [85–87]. Furthermore, due to its structure, the extracellular matrix alone can significantly contribute to the immobilization of heavy metals. The needle-shaped Ca-oxalate crystals in T. versicolor were mainly associated with older, more mature hyphae [88,89]. Although it seems to be proven that oxalate production decreases heavy metals toxicity for fungi, the relationships between oxalate contents and heavy metal resistance is not simple. In 19 strains of the brown-rot fungus Wolfiporia cocos, no relationship between amount of oxalic acid produced in liquid culture or wood and copper tolerance was found [90]. Another group of heavy metal-binding compounds produced by fungi are melanins, phenolic molecules associated with the cell wall. Some fungal melanins are efficient bioabsorbers of copper [91] or toxic tin compounds [92], and fungi produce them in response to copper [93]. An investigation of metal binding to the mycelial melanin of Armillaria spp. Rizzo et al. [94] found that the melanized rhizomorph mycelia concentrated Al, Zn, Fe, and Cu ions to 50–100 times the level found in surrounding soil. The concentrations of Al, Zn, Fe, Cu, and Pb ranged up to 3440, 1930, 1890, 15, and 680 ␮g g−1 , respectively. The metals were found only in the outer melanized part of rhizomorphs. Similar results were found with the melanized pseudosclerotial plates of P. weirii, that also accumulated heavy metals (Al, Ca, Cu, Fe, K, Mg, Mn, Na, Ni, Pb, Zn) from soil. The pseudosclerotial plates exposed to soil accumulated 15 ppm Cu, 940 ppm Fe, 64 ppm Mn, 7 ppm Ni, 29 ppm Pb, and 60 ppm Zn, whereas the control without contact with soil accumulated 4 ppm Cu, 8 ppm Fe, 43 ppm Mn, 1 ppm Ni, 3 ppm Pb, and 16 ppm Zn. In the case of all metals, the accumulation lead to higher metal concentrations in mycelium than in soil. Control mycelium of a brown-rot fungus Fomitopsis pinicola accumulated less metals than the pseudosclerotial plates [95]. In these cases, the metal ion coating of cell walls may play a role in the longevity and survival of the fungus in soil by protecting mycelia against antagonistic microorganisms or soil nematodes, perhaps because heavy metals inhibit the activity of excreted hydrolytic enzymes [94,96].

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It was noted that there is still no evidence for the production of phytochelatins or metallothioneins by white-rot fungi. Their role in the intracellular metal binding might be replaced with another types of peptide or protein molecules found in certain species. In P. ostreatus, copper and cadmium were found to be preferentially bound to low molecular weight compounds of 600 and 3500 Da, both compounds were also present in control mycelium. Amino acid analysis of these compounds revealed higher amounts of aspartic acid, cysteine and glycine [97]. A copper binding peptide with estimated MW of 2240 Da was purified from the fruit bodies of Grifola frondosa. Out of 23 amino acids, aspartic acid, glycine, glutamic acid and serine were the most abundant and no sulfur-containing amino acids were detected. It is not clear, if the peptide is induced by copper [98]. In D. quercina, induction of a 6000 Da protein was detected after addition of Cd. The concentration of this Cd-binding protein started to increase during the first hours after metal addition [99]. Synthesis of copper-binding compounds after exposure to Cu was also studied in P. chrysosporium, S. commune and P. ostreatus. Only in P. chrysosporium, did addition of Cu lead to formation of a metal-binding ligand, that was not present in the control. However, metal binding by the induced ligand was insignificant compared to other Cu-binding intracellular components [100]. It seems, that the role of intracellular binding peptides is limited in white-rot fungi compared to other groups of fungi and yeasts and that the extracellular and cell wall-associated binding is more important.

4. Ecology of metal–fungus interactions It is clear, that the interference of heavy metals with physiological, enzymatic and reproductive processes of white-rot fungi has its ecological consequences. The limitations in growth or reproduction in the presence of metals leads to the changes of community structure and the effects of heavy metals on enzymatic activities influences the energy flux in the ecosystem. It is therefore not surprising, that different species of white-rot fungi differ in the degree of their heavy metal tolerance. Sanglimsuwan et al. [8] studied 21 strains of 16 species of wood-rotting fungi for their resistance to metals. The minimum inhibitory concentrations were lowest in case of Hg (0.05–0.2 mM), Cd (0.5–5 mM), and Co (1–5 mM), and higher in case of Ni (0.7–7 mM), Zn (5–15 mM), and Cu (3–20 mM). The resistance differed from species to species with P. ostreatus and P. cystidosus being the most resistant. Among 15 wood-rotting species tested by Baldrian and Gabriel [24], S. hirsutum was the most Cd-tolerant, and the slow-growing Inonotus obliquus was the most sensitive. S. hirsutum was also the most Hg-tolerant species. In the case of mercury, increase of lag time was detected in addition to the reduction of radial growth rate [28]. Among the white-rot species tested by Palmans et al. [101], T. versicolor was

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found to be resistant to metals (Cd, Zn, Ni, Co, Cr, Mo, Pb, Hg, and Sn), whereas A. mellea, Lentinus sp., Pholiota nameko, Pleurotus sp., Pycnoporus sanguineus, and S. commune were clearly less resistant. Although copper is generally less toxic to white-rot fungi than Cd, it inhibited the growth of G. lucidum at concentrations less than 1 mM [27]. The differences in heavy metal tolerance occur also within strains of a single species. Major differences in Cu tolerance have been found among isolates of brown-rot fungi. Some of 36 strains of Anthrodia vaillantii tested were able to tolerate 40 mM Cu, although others were not able to grow at 3 mM Cu [102]. Similar results were obtained with W. cocos. Some isolates were able to decay pine wood treated with high concentrations of copper-based wood preservatives (CCA) [103,104]. High variability of growth response was also found in the case of Cd with another brown-rot fungus Piptoporus betulinus. Cd-tolerant as well as sensitive strains were found among 14 isolates from sites with different level of air pollution by cadmium [105]. The contents of heavy metals in fungal fruit bodies reflect the metal concentrations in their environment [106,107] and in several cases, metal-tolerant strains of basidiomycete fungi were isolated from contaminated sites [108,109]. In the ectomycorrhizal fungus Suillus luteus, isolates originating from heavy metal-polluted sites were significantly more tolerant to Cd and Zn (but not to Cu or Ni), than isolates from control sites [110]. However, in the wood-rotting fungus P. betulinus, no correlation between level of Cd-pollution of the atmosphere and resistance to Cd was found and even Cd-sensitive strains were isolated from sites with high pollution level [105]. On the other hand, laboratory experiments with Pleurotus florida and Pycnoporus sanguineus showed that it was possible to adapt the fungi to higher heavy metal concentrations [101]. The same was found in other white-rot fungi, including P. ostreatus and P. chrysosporium [111]. It seems, that there is no natural selection for metal-resistant strains at contaminated sites at least in fungi growing on wood. The situation might be different in soil, where the concentrations of the metals could be higher. The process of adaptation to metal stress is probably accompanied by the exclusion of metal-sensitive fungal species. This process is not limited only to the white-rot fungal species. Due to differences in metal tolerance of different microorganisms, the changes involve the whole soil microbial community structure. Addition of heavy metals leads also to the changes in the metabolic activity of microorganisms, including the decrease of ATP concentration and the soil respiration rate [112]. Laccase and cellulase activities can be found in forest litter [113,114] and these enzymes of white-rot fungi in addition to other enzymatic activities of soil microbiota contribute to substrate transformation and nutrient cycling in soils. The rate of transformation is modulated by heavy metals since these affect the activity of extracellular enzymes.

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5. Use of metals in antifungal compounds The fact that heavy metals are toxic to fungi has been widely used in the fight against fungal deterioration of materials including timber. Due to its high toxicity, the early preparations of biocides were based mostly on mercury. However, the toxicity of mercury to living organisms was also the case for ending the use of mercuric antifungals. The much less environmentally problematic copper was also found to exhibit good biocidal activity [115], but the major requirement of any formulation of copper-based wood preservative is efficacy against copper-tolerant fungi. The early reviews of preservative tolerance by Zabel and Cowling reported comparative tolerances of economically important decay fungi to current preservatives, most of them copper-based [116,117]. The fact, that there is a general correlation between tolerance of copper by decay fungi in wood and in agar [118] makes the testing of copper-based wood preservatives very simple. Among copper-containing wood protection agents, waterborne copper–chrome–arsenate (CCA) preservatives have established a world-wide reputation as the most versatile wood preservatives generally available. The first CCA preservative was patented in 1933 by the Indian worker Kamesan and since then, several other CCA preservatives have been developed. These vary in proportions and chemical forms of copper, chromium and arsenic compounds of which they are composed [119]. Chromated copper arsenate is currently a major commercial wood preservative for many applications in the US. In 1995, CCA represented more than 90% share of all water-borne preservatives [120]. In brown-rot fungi it was demonstrated, that fungi in general are not tolerant to both copper sulfate and arsenic pentoxide—the principal components of CCA [119]. White-rot fungi, e.g. T. versicolor were found to be even less resistant against CCA than all brown-rot species tested [121]. CCA retention of 4.1 kg m−3 reduced weight loss of Hevea brasiliensis wood caused by the white-rot fungi Irpex lacteus and T. versicolor to 8–10%. Retention of 14.5 kg m−3 prevented weight loss from exceeding 2%, whereas higher retentions were necessary for the treatment against the more tolerant brown-rot fungi [122]. CCA protected Acer rubrum sapwood against the same fungus even at 0.1% Cu w/w [123]. The use of CCA preservatives can locally pose a serious environmental problem due to leaching of the protectant and the toxicity of the resulting leachates [124]. Only recently, the use of wood preservatives based on CCA has become restricted in several countries due to environmental concerns. As a consequence, several chromium and arsenic-free wood preservatives, regarded as more environmentally acceptable, have been developed [125]. Newly developed copper-based wood preservatives combine copper with organic molecules. Copper naphthenatetreated wood of Quercus rubra was resistant to decay by white-rot fungi P. ostreatus and T. versicolor. At 1.28 kg m−3 Cu retention, the fungi caused less than 3% mass reduc-

tion within 16 weeks. Higher Cu contents was necessary to prevent decay by a copper-tolerant brown-rot fungus Poria placenta [126]. Only 2 kg m−3 Cu as waterborne copper naphthenate was sufficient for protection of Q. rubra and A. rubrum wood against white-rot fungi P. ostreatus, T. versicolor and I. lacteus [127]. Oilborne copper naphthenate was more effective than waterborne formulation against T. versicolor. With copper ethanolamine, 4 kg m−3 Cu was necessary for treatment of Q. rubra and A. rubrum wood against decay by white-rot fungi P. ostreatus, T. versicolor and I. lacteus, 4.5–5 kg m−3 was necessary for treatment against P. placenta [128]. Tannin–copper complexes were developed as wood preservatives with low toxicity that provide effective protection [129]. Tannin–copper complexes inhibited the growth of T. versicolor in a static liquid medium [130]. Other compounds found to be effective against T. versicolor decay include copper oxinate [131], copper-amine formulations [132] and fatty acid copper(II) carboxylates with nicotineamide, that stopped the growth of T. versicolor even at 10−3 M concentrations [133]. Even the treatment of wood with simple ammoniacal copper solutions to copper retention of 0.7 kg m−3 reduced weight loss to almost zero against T. versicolor [134]. Although white-rot fungi are usually less tolerant to copper-based wood preservatives than brown-rot fungi, T. versicolor and S. commune were able to degrade wood treated by copper sulfate and copper octanoate with ethanolamine, the preservatives effective against brown-rot fungi [135]. Organotin compounds have been utilized in a wood protection due to their efficacy against wood-destroying moulds and lower fungi. These compounds, bis-(tri-n-butyl-tin)oxide (TBTO) and n-butyl-tin-naphthenate (TBTN) found an important role after pentachlorophenol prohibition. 100 ppm TBTN or TBTA (tributyltin acetate) decreased the growth of T. versicolor to 35–38%. Among other derivatives tested, tri-n-butyl-tin-N-pyrrolidine dithiocarbamate and tri-n-butyl-tin-N,N-dipropyldithiocarbamate-propionate were the most effective. Organotin compounds are also effective against brown-rot fungi [136]. The research of new heavy metal-based antifungal compounds is still important, since there is a need for a formulation with good technological parameters (rapid timber impregnation) and the efficacy against wood-rotting fungi, due to environmental concerns.

6. Effects of metals on fungal biodegradation processes From biotechnological viewpoint, the occurrence of heavy metals poses a serious problem for the use of white-rot fungi to degrade persistent organic compounds including, e.g. synthetic textile dyes, PAHs or pesticides. Effluents from textile dyeing facilities contain metals used in dyes production technologies or in the molecule of textile dyes. Soil containing persistent organic compounds to be degraded by fungi is often contaminated by toxic levels of heavy metals.

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Since the effect of heavy metals on the activity of extracellular lignin-degrading enzymes has been described, it is clear that the presence of heavy metals affects also the biotechnological processes based on their activity. The decolorization of synthetic polymeric dye Poly R-478 by S. hirsutum was delayed and limited in the presence of 0.25 mM Cd in liquid mineral medium. In agar cultures, 0.1 mM Cd delayed the decolorization of Poly R-478 by S. hirsutum, P. chrysosporium, P. ostreatus, and P. cinnabarinus by 2–5 days compared to untreated controls. The presence of metals also delayed fungal colonization of agar plates [32]. Two subtropical strains of white-rot fungi, P. sanguineus and T. versicolor were able to decolorize dyes with different chemical structure at 0.25 mM Cd, Cu and Zn, whereas decolorization by P. chrysosporium was completely inhibited at concentrations as low as 0.1 mM. In all cases, decolorizing ability was more metal-sensitive than biomass production [137]. Also T. versicolor and P. sanguineus were able to decolorize Poly R-478 in the presence of heavy metals [101]. In the presence of more than 10 ppm CrO4 2− or 150 ppm Cr3+ , Bjerkandera sp. showed low decolorization of Direct Blue 69 and slow growth. During 15 days incubation, the fungus accumulated 30% chromium ions. Mn-peroxidase activity was not affected by the treatment [138]. In phtalocyanine dyes, heavy metals (Ni or Cu) are an inherent component of the molecule to be degraded. P. chrysosporium, B. adusta and T. versicolor effectively decolorized the Ni-containing Reactive blue 38 and Cu-containing Reactive blue 15. Ni contents in the liquid decreased during the process, whereas copper contents remained unchanged. At 200 ppm, the dyes were not toxic for fungi. The dyes could also be decolorized by crude preparations of extracellular enzymes [139]. P. chrysosporium was able to completely decolorize different Cu-containing phtalocyanine dyes in 200 ppm solution within 7 days. During the decolorization process, 50% of dye-bound copper was released into the supernatant. The toxicity of released metals was probably limited due to sorption to fungal biomass [140–142]. The levels of heavy metals encountered in polluted soils are usually several times higher than in waste waters. It was found, that the presence of copper in soil contaminated with PAHs decreased both microbial respiration and degradation of phenanthrene by native microflora. However, even at 700–7000 ppm Cu, the degradation slowly proceeded [143]. During the growth of P. ostreatus in nonsterile soil, activity of laccase was increased in soil containing 10–100 ppm Cd and slightly decreased at 500 ppm Cd. After 18 days of incubation, laccase activity reached the same values in all treatments. At 50–100 ppm Hg, laccase activity was delayed and the peak activity was decreased compared to control, but after 25 days, the activity in all treatments was the same [30]. Mn peroxidase was not significantly affected by Hg, except that in 50–100 ppm the early peak of activity on day 6 disappeared. In the presence of Cd, MnP activity was completely inhibited at 500 ppm. This was connected with a

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negligible degradation of PAHs by the fungus at 500 ppm Cd during 15-week cultivation. At other metal concentrations, the degradation of PAHs by the fungus was not substantially affected. The presence of 100–500 ppm Cd and 10–100 ppm Hg in soil increased the utilization of straw substrate by the culture. This was not found when fungus was absent. The presence of 100–500 ppm Cd and 10–100 ppm Hg also significantly decreased the colonization rate of P. ostreatus mycelium in nonsterile soil. The mycelium in higher metal concentration was only sparse, although the ligninolytic enzymes activities in treated and untreated soils were similar. At 100–500 ppm Cd and 50–100 ppm Hg, the fungus was not able to colonize a 70 mm layer of nonsterile soil [30]. In this particular case, even high concentrations of heavy metals did not result in the decrease of PAHs transformation. However, the reduction of colonization rate and incomplete colonization are major problems that cannot be simply overcome. Furthermore, the colonization of contaminated substrate is even more important under in situ conditions. The effect of heavy metals can also affect the degradation reactions by the regulation of other factors affecting biodegradation. The activation or inhibition of proteolytic enzymes by metals can change the turnover rate of extracellular enzymes [49] and several metals have been shown to affect hydrogen peroxide concentration in vitro. Hydrogen peroxide is one of the substrates for several lignin-degrading enzymes, but when produced in higher concentration it can inactivate Mn-peroxidase [144]. The information about the role of heavy metals in in situ degradation processes is still not completely clear and therefore needs further research.

7. Immobilization of metals using biosorption 7.1. Biosorption of heavy metals from solutions The use of microbial biomass for the biosorption of metals from industrial and municipal waste water has been proposed as a promising alternative to conventional heavy metal management strategies. Although the mechanism of metal sorption and uptake by microorganisms is still not completely understood, the sorption to polysaccharides, proteins or other molecules occurring in the outer layer of the cell wall probably plays the most important role. Experiments with chemically modified cell walls confirmed that various functional groups may participate in cation binding [145]. Wood-inhabiting basidiomycetes seem to be a promising material for biosorption since they can be easily cultivated in high yields on various substrates. Their pellets formed during submerged shaken cultivation have high surface/volume ratio and good mechanical properties. Their cell walls, similarly to other fungi consist mostly of polysaccharides, peptides and pigments that have a good capacity for heavy metals binding. It is therefore not surprising that during the last decade white-rot fungi have been tested for their ability

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Table 1 Biosorption of metals using white-rot fungi Mycelium Ganoderma lucidum G. lucidum G. lucidum Innonotus mikadoi Lepista nuda Oudemansiella mucida Phanerochaete chrysosporium P. chrysosporium P. chrysosporium P. chrysosporium P. chrysosporium P. chrysosporium P. chrysosporium P. chrysosporium P. chrysosporium P. chrysosporium P. chrysosporium P. chrysosporium Pleurotus ostreatus Pleurotus sajor-caju P. sajor-caju Pleurotus ostreiformis Pycnoporus cinnabarinus Pycnoporus sanguineus Trametes versicolor T. versicolor T. versicolor T. versicolor Tricholoma conglobatum Volvariella volvacea 8 species 12 species

Cu Cu Rare earth elements U, Th Cu Cu Cd, Cr, Ni, Pb Cd Cd Cu Cu Cd, Cr, Cu, Ni, Pb Hg, alkyl–Hg Pb Ni + Pb Cd, Cu, Pb Cd, Cu, Pb Pb Cu Cd, Cr, Ni, Pb Cd Cd, Cr, Ni, Pb Cu Pb, Cu, Cd Cd, Cr, Cu, Ni, Pb Ni Cd Cd U, Th Cd, Cr, Ni, Pb Cu Cd

[146,147] [148] [149] [150] [151] [151] [152] [153] [154] [155] [156] [157] [158] [159] [160] [161] [162] [163] [151] [152] [164] [152] [151] [165] [157] [166] [167] [153] [150] [152] [151] [153]

Immobilised mycelium P. chrysosporium P. sajor-caju T. versicolor T. versicolor

Hg Cd Cd Cd

[168] [169] [170] [171]

Fruit bodies Phellinus badius P. sanguineus 9 species

Pb Pb, Cu, Cd Cu

[172] [173] [174,175]

to adsorb heavy metals from solutions (Table 1). A broad range of heavy metals including rare earth elements, uranium and thorium were tested. In addition to mycelia from submerged cultivation, dried fruit bodies collected in nature and mycelia immobilized on various supports have been used. The adsorption of heavy metals to the mycelia of white-rot fungi fits the Langmuir adsorption isotherm. From the kinetic viewpoint it is a two-stage process with a rapid surface adsorption (30 min–1 h) and a slow intracellular diffusion (2–3 h). The equilibrium is reached within 6 h [153,159]. During the first minutes of interaction, pH decreases due to the release of protons [160,172]. The ion exchange mechanism can also play a role in metal binding. The sorption of Pb by P. sanguineus mycelia was accompanied by an ion exchange release of Ca from the mycelium [165]. Also the sorption of Cu on G. lucidum mycelium was based on

Ca2+ replacement [146]. However, ion exchange accounted only for 1% of Cu uptake by P. chrysosporium. The high capacity of copper uptake was mainly attributed to adsorption of very tiny colloids of copper hydroxide formed around pH 6, which was observed using SEM and indicated by pH effect on metal adsorption [156]. The ion exchange mechanism of metal binding makes the repeated cycles of sorption/desorption possible by altering the pH value [150,170,173]. Fungal cell wall has the key role in heavy metals sorption. The isolated cell wall fraction accounted for 38–77% of metal uptake and its sorption capacity was 20–50% higher than the overall binding capacity of the mycelium [151]. The heavy metal binding capacity is dependent on the mycelial age [159,164] and on the composition of culture media used for cultivation [151]. These phenomena are probably due to the changes in cell wall composition. Metal binding can also be affected by chemical or physical treatment of mycelia. These processes include acid and alkali treatment or the mode of drying of the mycelia. However, the effectiveness of such treatments depends on the metal and fungal species used [151,155,159,164]. Interspecific screening tests revealed, that white-rot fungal species differ in their absorption capacities. Fruit bodies of nine tropical wood-rotting fungi were tested for Cu biosorption by Muraleedharan et al. [175]. The maximum binding capacity differed in the range of 3 mg g−1 (T. lactenia) to 24 mg g−1 (G. lucidum). Pellets from submerged cultivation of 20 species of wood-rotting fungi, both white-rot and brown-rot were tested for Cd sorption by Gabriel et al. [153]. Metal accumulation ranged from 16 to 130 mg g−1 . The highest sorption capacity was found in F. pinicola (brown-rot, 130 mg g−1 ), T. versicolor (110 mg g−1 ) and P. chrysosporium (85 mg g−1 ). No differences were found between white-rot and brown-rot fungi. Under the same experimental conditions, cells of Saccharomyces cerevisiae accumulated only 6 mg g−1 Cd. Although several species of white-rot fungi have been tested for their sorption capacities, most work was performed using P. chrysosporium mycelia. The binding capacity for several metals ranges in tens of milligram per gram of dry weight (Table 2). Another advantage of the use of white-rot fungal mycelia for biosorption is the selectivity of different species for different metals. P. chrysosporium showed adsorptive capacities from equimolar solutions in the order Table 2 Maximum sorption capacities of P. chrysosporium mycelia with different metal ions Metal

Sorption capacity (mg g−1 dry weight)

Cd Cu Hg Ni Pb

110 60 61 56 108

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Cu > Pb > Cd [162], and for T. versicolor Pb > Ni > Cr > Cd > Cu [157]. Furthermore, the physical properties of mycelial pellets obtained using agitated cultivation of fungi provide a mechanically-stable material that can be used in different arrangements including fixed-bed column reactors [151,173]. However, the only industrial application of white-rot fungal mycelia until now was the use of G. lucidum in the removal of rare earth elements during monazite processing [149]. Recently, reports have appeared about the use of immobilized mycelia for metal sorption. The immobilization using Ca-alginate or carboxymethylcellulose further improves the mechanical stability of the sorbent and it has positive consequences for repeated use in cycles. The sorption capacity of these preparations is even better than the mycelia only, which could probably be due to the binding of metals to the carrier [168–171]. The technological use of white-rot fungal mycelia for biosorption seems promising from the technological viewpoint. However, there is a limitation in the costs of mycelia production, that is several times higher than in the case of waste yeast and filamentous fungal biomass waste from biotechnological processes.

treatment technologies. On the other hand, the understanding of the mechanism of heavy metals toxicity can offer novel formulations for antifungal compounds to be used in wood protection, that would selectively prevent fungal decay of timber without negative ecotoxicological consequences. Further research into metal–fungal relationships will therefore be a very good opportunity for the understanding of basic ecological concepts and the improvement or development of novel biotechnological processes like the application of fungal mycelia for selective removal of metals from polluted solutions or the regulation of in vitro enzymatic processes based on purified ligninolytic enzymes by metals.

7.2. Use of white-rot fungi for biomonitoring

References

As was shown, the wood-rotting fungi have a good potential to accumulate heavy metals from their environment. Since there are only very low concentrations of heavy metals in wood (except Zn), the main source for heavy metals in fruit bodies is the atmosphere. This has led to the use of wood-rotting fungi for the biomonitoring of atmosphere pollution. The results of such studies confirmed, that there is a clear relationship between air pollution and metal contents in fungal fruit bodies [107,176]. The biomonitoring is applicable for a wide range of heavy metals [177] and it provides a useful tool for environmental analysis.

8. Conclusions The interactions of white-rot fungi and toxic heavy metals have several physiological, ecological and technological consequences. The essential metals are necessary for fungal growth and development, but they are toxic when present in excess. The presence of heavy metals in the environment of fungi can lead to the changes in the structure and function of microbial communities in decaying wood or affect the decomposition process and nutrients turnover in soil. These processes as well as the mechanisms of active defense of fungi against metal toxicity are only partially understood and require more studies. The interaction of heavy metals with extracellular ligninolytic enzymes of white-rot fungi is particularly important for the understanding of the regulation of biotechnological processes of fungal degradation of xenobiotics. The presence of heavy metals on sites with mixed contamination can be a serious limitation for in situ

Acknowledgments This work was supported by the Grant Agency of the Czech Republic (204/02/P100), by the Grant Agency of the Czech Academy of Sciences (B5020202) and by the Institutional Research Concept no. AV0Z5020903 of the Institute of Microbiology, ASCR.

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