Arch Microbiol DOI 10.1007/s00203-014-0993-z
Original Paper
Fungal accumulation of metals from building materials during brown rot wood decay Anne Christine Steenkjær Hastrup · Bo Jensen · Jody Jellison
Received: 20 February 2014 / Revised: 30 April 2014 / Accepted: 10 May 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract This study analyzes the accumulation and translocation of metal ions in wood during the degradation performed by one strain of each of the three brown rot fungi; Serpula lacrymans, Meruliporia incrassata and Coniophora puteana. These fungi species are inhabitants of the built environment where the prevention and understanding of fungal decay is of high priority. This study focuses on the influence of various building materials in relation to fungal growth and metal uptake. Changes in the concentration of iron, manganese, calcium and copper ions in the decayed wood were analyzed by induced coupled plasma spectroscopy and related to wood weight loss and oxalic acid accumulation. Metal transport into the fungal inoculated wood was found to be dependent on the individual strain/species. The S. lacrymans strain caused a significant increase in total iron whereas the concentration of copper ions in the wood appeared decreased after 10 weeks of decay. Wood inoculated with the M. incrassata isolate
showed the contrary tendency with high copper accumulation and low iron increase despite similar weight losses for the two strains. However, significantly lower oxalic acid accumulation was recorded in M. incrassata degraded wood. The addition of a building material resulted in increased weight loss in wood degraded by C. puteana in the soil-block test; however, this could not be directly linked specifically to the accumulation of any of the four metals recorded. The accumulation of oxalic acid seemed to influence the iron uptake. The study assessing the influence of the presence of soil and glass in the soil-block test revealed that soil contributed the majority of the metals for uptake by the fungi and contributed to increased weight loss. The varying uptake observed among the three brown rot fungi strains toward the four metals analyzed may be related to the specific non-enzymatic and enzymatic properties including bio-chelators employed by each of the species during wood decay.
Communicated by Erko Stackebrandt.
Keywords Wood decay · Brown rot · Oxalic acid · Metal accumulation · HPLC · ICP
A. C. S. Hastrup Wood and Bio Based Materials, Danish Technological Institute, Gregersensvej 1, 2630 Taastrup, Denmark A. C. S. Hastrup (*) · J. Jellison Study Performed at School of Biology and Ecology, University of Maine, 311 Hitchner Hall, Orono, ME 04469, USA e-mail: [email protected] B. Jensen Molecular Microbial Ecology Group, University of Copenhagen, Universitetsparken 15 Building 1, 2100 Copenhagen, Denmark J. Jellison Department of Plant Pathology, Physiology, Weed Science, Virginia Tech, 104‑C Hutcheson Hall, Blacksburg, VA 24061, USA
Introduction Metal ions are involved in all aspects of microbial growth and metabolism. They are actively accumulated to varying extents in wood during decay by brown rot fungi (Ostrofsky et al. 1997; Schilling and Bissonnette 2008; Schilling and Jellison 2006). The metal ions are solubilized from mineral materials, in particular from lithological sources in the surrounding environment, translocated through hyphal cords and used for specific functions in the fungi (Connolly et al. 1999). The importance of a few metals such as iron, calcium, manganese and copper has been
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particularly investigated with regard to fungal growth and decay. Iron is the most abundant metal on earth, but exists in nature and in the aerobic environments of wood predominantly in the ferric (III) oxidation state (Arantes et al. 2012; Goodell et al. 1997). The low iron content in wood of 0.18 μmol/g ± 0.1 and the tight association to the cell wall makes it not readily available for fungal assimilation (Jellison et al. 1992). Iron is crucial for the brown rot fungi due to the involvement in Fenton chemistry (Goodell et al. 1997; Hastrup et al. 2011). The Fenton reaction is the catalyzed decomposition of dilute hydrogen peroxide (H2O2) by iron (II) to form hydroxyl radicals. These highly reactive oxygen species are generally accepted to be part of the non-enzymatic decay process, which facilitates the initial structural changes in the wood leading to increased porosity and facilitation of enzymatic decay (Goodell et al. 1997; Hammel et al. 2002; Koenigs 1974; Tanaka et al. 2007). Fungal production and accumulation of oxalic acid in early stages of decay causes a rapid decline in pH in the immediate environment around the hyphae to approximately pH ~2–3 (Green et al. 1991; Hyde and Wood 1997). Oxalic acid thus facilitates iron solubilization, complexing and additional allocation of iron from the surrounding environment (Arantes et al. 2009; Goodell et al. 1997) and is therefore regarded as a metal transporter (Arantes et al. 2009; Goodell et al. 1997; Kerem et al. 1999). The acidic environment obtained by the accumulation of oxalic acid generates suitable conditions for the Fenton reaction as iron (II) will be present as free Fe2+-ion or in an iron-oxalate complex and autoxidation of Fe2+–Fe3+ is avoided (Contreras et al. 2007). Manganese is another such transition element that exists naturally in nature in the (+II) and (+IV) oxidation states, although other valence states, such as (+III) and (+VII), also occur (Henry 2003; Watts et al. 2005). For this reason, manganese has been suggested as a possible substitute for iron in a Fenton-like reaction (Illman et al. 1988a, b). Watts et al. (2005) found that soluble manganese and manganese oxides catalyze hydrogen peroxide decomposition, albeit at a very low rate at acidic pH values ( 0.1). The accumulated amount of calcium corresponds with other studies on fungal degraded wood showing an increase in calcium content during decay (Schilling 2010; Schmidt 2007). The ability by these fungi to form cords can be a large factor in the
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translocation and accumulation of calcium (Connolly and Jellison 1995). Wood inoculated with M. incrassata and incubated with mortar showed significantly higher calcium accumulation compared to degraded wood incubated with gypsum board and inoculated control blocks (p = 0.022) (Fig. 1). The presence of glass fiber also resulted in high calcium uptake (p = 0.063). The lower calcium concentration in gypsum board incubated wood blocks was not correlated with a lower weight loss; however, it corresponded to a lower accumulation of oxalic acid in this group compared to mortar and glass fiber exposed blocks. However, this factor could not explain the lower accumulation of calcium in the internal control of M. incrassata-degraded wood with no additional calcium source. The calcium accumulation was not correlated with the calcium content in the added building material supplements despite a higher concentration in some products compared to others: Soil = 0.018 g Ca g−1 in 2 g, glass fiber = 0.14 g Ca g−1 in 0.7 g, gypsum board = 0.3 g Ca g−1 in 4 g and mortar = 0.5 g Ca g−1 in 12 g. Further, the analyses did not show a higher uptake of calcium in S. lacrymans-degraded wood blocks compared to the two other brown rot fungi strains despite a higher concentration of oxalic acid. This contradicts the suggestion that S. lacrymans is specific in its needs for calcium to neutralize the accumulated oxalic acid (Bech-Andersen 1987). Instead a maximum accumulation of calcium in the degraded wood was found to be around 20–25 μ mol g−1 initial weights for all three fungal species despite varying availability of calcium in the growth medium and varying oxalic acid accumulation. Manganese uptake The uptake of manganese was low in both S. lacrymans and C. puteana, which corresponds with previous reports of Fe accumulation but no Mn uptake in the wood despite of the two metals having roughly similar soil availabilities (Connolly and Jellison 1995). However, M. incrassata caused significant uptake of Mn in wood blocks inoculated with gypsum board and in the control samples (p = 0.001, p = 0.000, respectively) (Fig. 2). The same was observed in wood incubated in polystyrene jars and inoculated with this fungus, where addition of gypsum board resulted in a significant accumulation of Mn (1.57 ± 0.21 μmol g−1). The presence of soil resulted in a final higher Mn concentration in three out of the five wood blocks (0.72 ± 0.67 μmol g−1). Mortar and jar glass did not cause any noteworthy uptake (0.21 ± 0.04 μmol g−1 and 0.17 ± 0.02 μmol g−1, respectively). The wood blocks grown with glass fiber showed considerable variation with 4 out of 6 replicate samples showing no uptake of Mn (0.58 ± 0.58 μmol g−1). Wood blocks inoculated in the presence of gypsum board and soil in the polystyrene jars
Arch Microbiol Fig. 2 Accumulation of manganese ions (μmol g−1 initial wood weight) in wood blocks degraded by the three brown rot fungi S. lacrymans, C. puteana and M. incrassata
1,8 1,6 1,4
µmol/g
1,2 1,0 0,8 0,6 0,4 0,2
Fig. 3 Copper accumulation in wood blocks inoculated with brown rot fungi for 10 weeks (μmol g−1 initial weight)
M. incrassata
Gypsum board
Glass fiber
Mortar
Int. Control
Glass fiber
Mortar
Int. Control
C. puteana
Gypsum board
S. lacrymans
Gypsum board
Glass fiber
Mortar
Int. Control
No fungi
-
0,18 0,16 0,14
µmol/g
0,12 0,10 0,08 0,06 0,04 0,02
S. lacrymans
were the two only sample groups out of the six different setups in this polystyrene jar test that showed weight loss of more than 10 % after 10 weeks of decay. The accumulation of Mn by M. incrassata could be a substitution for iron. The role of manganese in the biodegradation of wood by brown rot wood decay fungi is less elucidated than in white rot fungi where the metal plays a bigger role due to the manganese-requiring lignin-degrading enzyme manganese peroxidase. Only few brown rot fungi have been found to produce Mn peroxidase (Dey et al. 1994; Huang et al. 2009; Ruiz-Dueñas et al. 2013). Still, it is ambiguous if peroxidases are expressed in brown rot fungi and what their role are in the brown rot lignin modification process (Morgenstern et al. 2010). Complementary, divalent manganese is mobilized by P. placenta and G. trabeum during degradation of wood (Illman et al. 1988a, 1989). Thus, due to the
C. puteana
Gypsum board
Glass fiber
Mortar
Int. Control
Gypsum board
Glass fiber
Mortar
Int. Control
Gypsum board
Glass fiber
Mortar
Int. Control
No fungi
-
M. incrassata
redox potential of manganese and the possible substitution of iron, manganese could have a role in the non-enzymatic mechanisms involved in wood biodegradation (Hastrup et al. 2011; Illman and Highley 1989; Jellison et al. 1992, 1997). Copper uptake The recorded copper levels in the wood before but also after fungal inoculation, growth and decay are low, which can be critical for the interpretation. C. puteana and M. incrassata both induced significant accumulation of copper (p = 0.001, p = 0.000, respectively). However, wood incubated with S. lacrymans appeared reduced in copper content after 10 weeks of decay compared to the control wood, although this was not significant (p = 0.537) (Fig. 3). The copper ions may be translocated away from the wood via
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the mycelium hyphae to a storage located outside the wood, such as the soil, through mycelial cords (Connolly and Jellison 1997; Schilling and Jellison 2004). Alternatively, they may be complexed into copper oxalate crystals and deposited on the hyphae away from the wood, i.e., in the soil or on the building material supplements (Hastrup et al. 2012). The fluctuation in the metal content in the soil or the building/metal supplement following decay was unfortunately not recorded to validate this theory. The degree of degradation caused by the C. puteana strain seemed to influence the copper accumulation or vice versa despite correction for weight loss, although with no statistical differences observed between the four treatment groups (p > 0.16). This was also observed for iron and calcium accumulation. No correlation was found between the accumulation of oxalic acid in the C. puteana strain and the uptake of copper. The accumulation of copper in M. incrassata-degraded wood was associated with the level of oxalic acid in the wood blocks but not the decay rate (Table 1). This resulted in a lower concentration of copper in wood from jars supplemented with glass fiber and gypsum, although not significantly (p = 0.357, p = 0.059) compared to samples from jars with no added building components (internal control), which had a higher oxalic acid concentration. The copper levels in the soil or in the building materials did not reach toxic levels (Clausen and Green 2003; Hastrup et al. 2005a). The copper tolerance or requirement in the M. incrassata strain may be different from the strains of the two other brown rot species and the translocation might be accompanying by something other than oxalic acid.
Conclusion The ICP analysis showed high variability among the three fungal strains of the three brown rot species in their efficiency, preference and/or need for metal uptake during wood decay. The S. lacrymans strain caused an elevated iron uptake relative to the other two, taxonomically closely related species, whereas M. incrassata caused accumulation of manganese in two out of the four treatments. Copper was found in highest amount in C. puteana and M. incrassata-degraded wood. Calcium accumulation was observed in wood degraded by all three fungi although not in all treatment groups. The variation among the three brown rot fungi strains analyzed in the preferences toward the various metals analyzed may be related to the distinct non-enzymatic and enzymatic properties employed during growth and decay by each of the strains/species. A part of the variation was allocated the amount of soluble and total extractable oxalic acid accumulated. The regulation of oxalic acid can be
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Arch Microbiol
related to iron and copper translocation in the wood blocks during decay. Other mechanisms such as fungal iron-chelators, cellulolytic enzymes, oxalic acid carboxylation and decarboxylation or the production of hydrogen peroxide are further parameters causing variance in uptake or translocation of the metals tested. Acknowledgments The authors thank Joan Perkins for valuable help with the ICP analysis. Caitlin L. Howell for assistance with statistical analysis. Jonathan Schilling for critical review and comments on the manuscript. The work was supported by Frimodt-Heineke Fonden, Ingeniør Svend G. Fiedler og Hustrus legat, and by the University of Maine.
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(eds) Anonymous Biodeterioration Research 2. Springer, US, pp 485–496 Jarosz-Wilkolazka A, Gadd GM (2003) Oxalate production by woodrotting fungi growing in toxic metal-amended medium. Chemosphere 52:541–547 Jellison J, Smith K, Shortle W (1992) Cation analysis of wood degraded by white-and brown-rot fungi. In: International Research Group on Wood PreservationDocument IRG/WP/92-1552 Jellison J, Connolly J, Goodell B, Doyle B, Illman B, Fekete F, Ostrofsky A (1997) The role of cations in the biodegradation of wood by the brown rot fungi. Int Biodeterior Biodegrad 39:165–179 Kerem Z, Jensen KA, Hammel KE (1999) Biodegradative mechanism of the brown rot basidiomycete Gloeophyllum trabeum: evidence for an extracellular hydroquinone-driven Fenton reaction. FEBS Lett 446:49–54 Koenig JW (1974) Hydrogen peroxide and iron: a proposed system for decomposition of wood by brown-rot basidiomycetes. Wood Fiber Sci 6:66–80 Lebow S (2004) Alternatives to chromated copper arsenate (CCA) for residential construction. Environmental impacts of preservativetreated wood conference Orlando, Florida, USA Magro P, Marciano P, Di Lenna P (1984) Oxalic acid production and its role in pathogenesis of Sclerotinia sclerotiorum. FEMS Microbiol Lett 24:9–12 Micales JA (1992) Oxalic acid metabolism of Postia placenta. In: International Research Group on Wood PreservationDocument IRG/WP/92-1556 Morgenstern I, Robertson DL, Hibbett DS (2010) Characterization of Three mnp Genes of Fomitiporia mediterranea and report of additional class II peroxidases in the order Hymenochaetales. Appl Environ Microbiol 76(19):6431–6440 Morris P (1992) Available iron promotes brown rot of treated wood. In: International Research Group on Wood PreservationDocument IRG/WP/92-1526 Munir E, Yoon JJ, Tokimatsu T, Hattori T, Shimada M (2001) New role for glyoxylate cycle enzymes in wood-rotting basidiomycetes in relation to biosynthesis of oxalic acid. J Wood Sci 47:368–373 Murphy R, Levy J (1983) Production of copper oxalate by some copper tolerant fungi. Trans Br Mycol Soc 81:165–168 Nicholas D, Schultz T (1997) Comparative performance of several ammoniacal copper preservative systems. In: International Research Group on Wood PreservationDocument IRG/WP/97-30151 Ostrofsky A, Jellison J, Smith K, Shortle W (1997) Changes in cation concentrations in red spruce wood decayed by brown rot and white rot fungi. Can J Forest Res 27:567–571 Paajanen L (1993) Iron promotes decay capacity of Serpula lacrymans. In: International Research Group on Wood PreservationDocument IRG/WP/93-10008 Qiu Q, Kumosa M (1997) Corrosion of E-glass fibers in acidic environments. Compos Sci Technol 57:497–507 Ruiz-Dueñas FJ, Lundell T, Floudas D, Nagy LG, Barrasa JM, Hibbett DS, Martínez AT (2013) Lignin-degrading peroxidases in polyporales: an evolutionary survey based on 10 sequenced genomes. Mycologia 105(6):1428–1444 Sayer JA, Gadd GM (1997) Solubilization and transformation of insoluble inorganic metal compounds to insoluble metal oxalates by Aspergillus niger. Mycol Res 101:653–661 Schilling JS (2010) Effects of calcium-based materials and iron impurities on wood degradation by the brown rot fungus Serpula lacrymans. Holzforschung 64:93–99 Schilling JS, Bissonnette KM (2008) Iron and calcium translocation from pure gypsum and iron-amended gypsum by two brown rot fungi and a white rot fungus. Holzforschung 62:752–758
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Original Paper
Fungal accumulation of metals from building materials during brown rot wood decay Anne Christine Steenkjær Hastrup · Bo Jensen · Jody Jellison
Received: 20 February 2014 / Revised: 30 April 2014 / Accepted: 10 May 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract This study analyzes the accumulation and translocation of metal ions in wood during the degradation performed by one strain of each of the three brown rot fungi; Serpula lacrymans, Meruliporia incrassata and Coniophora puteana. These fungi species are inhabitants of the built environment where the prevention and understanding of fungal decay is of high priority. This study focuses on the influence of various building materials in relation to fungal growth and metal uptake. Changes in the concentration of iron, manganese, calcium and copper ions in the decayed wood were analyzed by induced coupled plasma spectroscopy and related to wood weight loss and oxalic acid accumulation. Metal transport into the fungal inoculated wood was found to be dependent on the individual strain/species. The S. lacrymans strain caused a significant increase in total iron whereas the concentration of copper ions in the wood appeared decreased after 10 weeks of decay. Wood inoculated with the M. incrassata isolate
showed the contrary tendency with high copper accumulation and low iron increase despite similar weight losses for the two strains. However, significantly lower oxalic acid accumulation was recorded in M. incrassata degraded wood. The addition of a building material resulted in increased weight loss in wood degraded by C. puteana in the soil-block test; however, this could not be directly linked specifically to the accumulation of any of the four metals recorded. The accumulation of oxalic acid seemed to influence the iron uptake. The study assessing the influence of the presence of soil and glass in the soil-block test revealed that soil contributed the majority of the metals for uptake by the fungi and contributed to increased weight loss. The varying uptake observed among the three brown rot fungi strains toward the four metals analyzed may be related to the specific non-enzymatic and enzymatic properties including bio-chelators employed by each of the species during wood decay.
Communicated by Erko Stackebrandt.
Keywords Wood decay · Brown rot · Oxalic acid · Metal accumulation · HPLC · ICP
A. C. S. Hastrup Wood and Bio Based Materials, Danish Technological Institute, Gregersensvej 1, 2630 Taastrup, Denmark A. C. S. Hastrup (*) · J. Jellison Study Performed at School of Biology and Ecology, University of Maine, 311 Hitchner Hall, Orono, ME 04469, USA e-mail: [email protected] B. Jensen Molecular Microbial Ecology Group, University of Copenhagen, Universitetsparken 15 Building 1, 2100 Copenhagen, Denmark J. Jellison Department of Plant Pathology, Physiology, Weed Science, Virginia Tech, 104‑C Hutcheson Hall, Blacksburg, VA 24061, USA
Introduction Metal ions are involved in all aspects of microbial growth and metabolism. They are actively accumulated to varying extents in wood during decay by brown rot fungi (Ostrofsky et al. 1997; Schilling and Bissonnette 2008; Schilling and Jellison 2006). The metal ions are solubilized from mineral materials, in particular from lithological sources in the surrounding environment, translocated through hyphal cords and used for specific functions in the fungi (Connolly et al. 1999). The importance of a few metals such as iron, calcium, manganese and copper has been
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particularly investigated with regard to fungal growth and decay. Iron is the most abundant metal on earth, but exists in nature and in the aerobic environments of wood predominantly in the ferric (III) oxidation state (Arantes et al. 2012; Goodell et al. 1997). The low iron content in wood of 0.18 μmol/g ± 0.1 and the tight association to the cell wall makes it not readily available for fungal assimilation (Jellison et al. 1992). Iron is crucial for the brown rot fungi due to the involvement in Fenton chemistry (Goodell et al. 1997; Hastrup et al. 2011). The Fenton reaction is the catalyzed decomposition of dilute hydrogen peroxide (H2O2) by iron (II) to form hydroxyl radicals. These highly reactive oxygen species are generally accepted to be part of the non-enzymatic decay process, which facilitates the initial structural changes in the wood leading to increased porosity and facilitation of enzymatic decay (Goodell et al. 1997; Hammel et al. 2002; Koenigs 1974; Tanaka et al. 2007). Fungal production and accumulation of oxalic acid in early stages of decay causes a rapid decline in pH in the immediate environment around the hyphae to approximately pH ~2–3 (Green et al. 1991; Hyde and Wood 1997). Oxalic acid thus facilitates iron solubilization, complexing and additional allocation of iron from the surrounding environment (Arantes et al. 2009; Goodell et al. 1997) and is therefore regarded as a metal transporter (Arantes et al. 2009; Goodell et al. 1997; Kerem et al. 1999). The acidic environment obtained by the accumulation of oxalic acid generates suitable conditions for the Fenton reaction as iron (II) will be present as free Fe2+-ion or in an iron-oxalate complex and autoxidation of Fe2+–Fe3+ is avoided (Contreras et al. 2007). Manganese is another such transition element that exists naturally in nature in the (+II) and (+IV) oxidation states, although other valence states, such as (+III) and (+VII), also occur (Henry 2003; Watts et al. 2005). For this reason, manganese has been suggested as a possible substitute for iron in a Fenton-like reaction (Illman et al. 1988a, b). Watts et al. (2005) found that soluble manganese and manganese oxides catalyze hydrogen peroxide decomposition, albeit at a very low rate at acidic pH values ( 0.1). The accumulated amount of calcium corresponds with other studies on fungal degraded wood showing an increase in calcium content during decay (Schilling 2010; Schmidt 2007). The ability by these fungi to form cords can be a large factor in the
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translocation and accumulation of calcium (Connolly and Jellison 1995). Wood inoculated with M. incrassata and incubated with mortar showed significantly higher calcium accumulation compared to degraded wood incubated with gypsum board and inoculated control blocks (p = 0.022) (Fig. 1). The presence of glass fiber also resulted in high calcium uptake (p = 0.063). The lower calcium concentration in gypsum board incubated wood blocks was not correlated with a lower weight loss; however, it corresponded to a lower accumulation of oxalic acid in this group compared to mortar and glass fiber exposed blocks. However, this factor could not explain the lower accumulation of calcium in the internal control of M. incrassata-degraded wood with no additional calcium source. The calcium accumulation was not correlated with the calcium content in the added building material supplements despite a higher concentration in some products compared to others: Soil = 0.018 g Ca g−1 in 2 g, glass fiber = 0.14 g Ca g−1 in 0.7 g, gypsum board = 0.3 g Ca g−1 in 4 g and mortar = 0.5 g Ca g−1 in 12 g. Further, the analyses did not show a higher uptake of calcium in S. lacrymans-degraded wood blocks compared to the two other brown rot fungi strains despite a higher concentration of oxalic acid. This contradicts the suggestion that S. lacrymans is specific in its needs for calcium to neutralize the accumulated oxalic acid (Bech-Andersen 1987). Instead a maximum accumulation of calcium in the degraded wood was found to be around 20–25 μ mol g−1 initial weights for all three fungal species despite varying availability of calcium in the growth medium and varying oxalic acid accumulation. Manganese uptake The uptake of manganese was low in both S. lacrymans and C. puteana, which corresponds with previous reports of Fe accumulation but no Mn uptake in the wood despite of the two metals having roughly similar soil availabilities (Connolly and Jellison 1995). However, M. incrassata caused significant uptake of Mn in wood blocks inoculated with gypsum board and in the control samples (p = 0.001, p = 0.000, respectively) (Fig. 2). The same was observed in wood incubated in polystyrene jars and inoculated with this fungus, where addition of gypsum board resulted in a significant accumulation of Mn (1.57 ± 0.21 μmol g−1). The presence of soil resulted in a final higher Mn concentration in three out of the five wood blocks (0.72 ± 0.67 μmol g−1). Mortar and jar glass did not cause any noteworthy uptake (0.21 ± 0.04 μmol g−1 and 0.17 ± 0.02 μmol g−1, respectively). The wood blocks grown with glass fiber showed considerable variation with 4 out of 6 replicate samples showing no uptake of Mn (0.58 ± 0.58 μmol g−1). Wood blocks inoculated in the presence of gypsum board and soil in the polystyrene jars
Arch Microbiol Fig. 2 Accumulation of manganese ions (μmol g−1 initial wood weight) in wood blocks degraded by the three brown rot fungi S. lacrymans, C. puteana and M. incrassata
1,8 1,6 1,4
µmol/g
1,2 1,0 0,8 0,6 0,4 0,2
Fig. 3 Copper accumulation in wood blocks inoculated with brown rot fungi for 10 weeks (μmol g−1 initial weight)
M. incrassata
Gypsum board
Glass fiber
Mortar
Int. Control
Glass fiber
Mortar
Int. Control
C. puteana
Gypsum board
S. lacrymans
Gypsum board
Glass fiber
Mortar
Int. Control
No fungi
-
0,18 0,16 0,14
µmol/g
0,12 0,10 0,08 0,06 0,04 0,02
S. lacrymans
were the two only sample groups out of the six different setups in this polystyrene jar test that showed weight loss of more than 10 % after 10 weeks of decay. The accumulation of Mn by M. incrassata could be a substitution for iron. The role of manganese in the biodegradation of wood by brown rot wood decay fungi is less elucidated than in white rot fungi where the metal plays a bigger role due to the manganese-requiring lignin-degrading enzyme manganese peroxidase. Only few brown rot fungi have been found to produce Mn peroxidase (Dey et al. 1994; Huang et al. 2009; Ruiz-Dueñas et al. 2013). Still, it is ambiguous if peroxidases are expressed in brown rot fungi and what their role are in the brown rot lignin modification process (Morgenstern et al. 2010). Complementary, divalent manganese is mobilized by P. placenta and G. trabeum during degradation of wood (Illman et al. 1988a, 1989). Thus, due to the
C. puteana
Gypsum board
Glass fiber
Mortar
Int. Control
Gypsum board
Glass fiber
Mortar
Int. Control
Gypsum board
Glass fiber
Mortar
Int. Control
No fungi
-
M. incrassata
redox potential of manganese and the possible substitution of iron, manganese could have a role in the non-enzymatic mechanisms involved in wood biodegradation (Hastrup et al. 2011; Illman and Highley 1989; Jellison et al. 1992, 1997). Copper uptake The recorded copper levels in the wood before but also after fungal inoculation, growth and decay are low, which can be critical for the interpretation. C. puteana and M. incrassata both induced significant accumulation of copper (p = 0.001, p = 0.000, respectively). However, wood incubated with S. lacrymans appeared reduced in copper content after 10 weeks of decay compared to the control wood, although this was not significant (p = 0.537) (Fig. 3). The copper ions may be translocated away from the wood via
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the mycelium hyphae to a storage located outside the wood, such as the soil, through mycelial cords (Connolly and Jellison 1997; Schilling and Jellison 2004). Alternatively, they may be complexed into copper oxalate crystals and deposited on the hyphae away from the wood, i.e., in the soil or on the building material supplements (Hastrup et al. 2012). The fluctuation in the metal content in the soil or the building/metal supplement following decay was unfortunately not recorded to validate this theory. The degree of degradation caused by the C. puteana strain seemed to influence the copper accumulation or vice versa despite correction for weight loss, although with no statistical differences observed between the four treatment groups (p > 0.16). This was also observed for iron and calcium accumulation. No correlation was found between the accumulation of oxalic acid in the C. puteana strain and the uptake of copper. The accumulation of copper in M. incrassata-degraded wood was associated with the level of oxalic acid in the wood blocks but not the decay rate (Table 1). This resulted in a lower concentration of copper in wood from jars supplemented with glass fiber and gypsum, although not significantly (p = 0.357, p = 0.059) compared to samples from jars with no added building components (internal control), which had a higher oxalic acid concentration. The copper levels in the soil or in the building materials did not reach toxic levels (Clausen and Green 2003; Hastrup et al. 2005a). The copper tolerance or requirement in the M. incrassata strain may be different from the strains of the two other brown rot species and the translocation might be accompanying by something other than oxalic acid.
Conclusion The ICP analysis showed high variability among the three fungal strains of the three brown rot species in their efficiency, preference and/or need for metal uptake during wood decay. The S. lacrymans strain caused an elevated iron uptake relative to the other two, taxonomically closely related species, whereas M. incrassata caused accumulation of manganese in two out of the four treatments. Copper was found in highest amount in C. puteana and M. incrassata-degraded wood. Calcium accumulation was observed in wood degraded by all three fungi although not in all treatment groups. The variation among the three brown rot fungi strains analyzed in the preferences toward the various metals analyzed may be related to the distinct non-enzymatic and enzymatic properties employed during growth and decay by each of the strains/species. A part of the variation was allocated the amount of soluble and total extractable oxalic acid accumulated. The regulation of oxalic acid can be
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Arch Microbiol
related to iron and copper translocation in the wood blocks during decay. Other mechanisms such as fungal iron-chelators, cellulolytic enzymes, oxalic acid carboxylation and decarboxylation or the production of hydrogen peroxide are further parameters causing variance in uptake or translocation of the metals tested. Acknowledgments The authors thank Joan Perkins for valuable help with the ICP analysis. Caitlin L. Howell for assistance with statistical analysis. Jonathan Schilling for critical review and comments on the manuscript. The work was supported by Frimodt-Heineke Fonden, Ingeniør Svend G. Fiedler og Hustrus legat, and by the University of Maine.
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