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Attenuated total reflection infrared spectroscopy of white-rot decayed beech wood
ARTICLE IN PRESS
International Biodeterioration & Biodegradation 55 (2005) 247–251 www.elsevier.com/locate/ibiod
Attenuated total reflection infrared spectroscopy of white-rot decayed beech wood Behbood Mohebby Department of Wood and Paper Sciences, Faculty of Natural Resources and Marine Sciences, Tarbiat Modarress University, P.O. Box 46414-356, Noor, Iran Received 4 August 2004; received in revised form 13 November 2004; accepted 29 December 2004
Abstract Small blocks of beech wood were exposed to the white-rot fungus Trametes versicolor for a period of 84 days to investigate chemical alteration in decayed wood by infrared spectroscopy. Decayed samples were analyzed at 2 week intervals by using attenuated total teflection (ATR) infrared spectroscopy as a rapid method. Analyses showed that chemical alteration in wood began after the second week of exposure. The appearance of new peaks indicated chemical modification of cell walls between days 28 and 70 of exposure to the fungus, and the disappearance of the peaks at day 84 indicates removal of the cell wall constituents. This investigation showed that ATR spectroscopy is a very applicable and rapid method for studying wood biodegradation. r 2005 Elsevier Ltd. All rights reserved. Keywords: Attenuated total reflection infrared spectroscopy; Beech; White-rot decay; Trametes versicolor
1. Introduction Several types of biodegradation have been recognized in wood, viz. fungal decay, bacterial degradation and insect attack. Fungal decay is the most important and widespread type of degradation and is caused by white-, brown- or soft-rot fungi (Zabell and Morrell, 1992; Eriksson et al., 1990). Different methods and techniques have been applied to monitor fungal decay, including gravimetric analysis (see, for example, Wa¨lchli, 1970); loss of dynamic modulus of elasticity (Machek et al., 1998a, b; Mohebby and Militz, 2002; Mohebby, 2003); technical microscopy, e.g. light, polarized and electron microscopies (Anagnost, 1998); NMR and FTIR spectroscopy (Pandey and Pitman, 2003); microcalorimetry (Xie et al., 1997; Bjurman and Wadso¨, 2000; Mohebby, 2003; Mohebby et al., 2003), ergosterol assay (Mohebby, 2003; Mohebby et al., 2003); analysis of flourescein diacetate (FDA) (Mohebby, 2003); GC–MS spectroscopy; chemical analysis (Zabell and Morrell, E-mail address: [email protected]. 0964-8305/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2005.01.003
1992; Mohebby and Militz, 2002); electronic nose (Nilsson, 1998); electron paramagnetic resonance and synchrotron X-ray fluorescence (Illman and Bajt, 1997); immunodiagnostics (Clausen, 1996; Clausen and Green, 1998); bending vibration technique (Ouis, 2000); resistography (Rinn et al., 1996); and scanning technique (Ross and de Groot, 1998). White-, brown- and soft-rot fungi attack wood based on their enzymatic systems; these enzymes penetrate the wood cell wall, alter its chemistry and break down the cell-wall polymers into constituents that can be taken up by hyphae. Brown-rot fungi selectively decay cell-wall polysaccharides, with limited lignin degradation. The decay system in this type is based on both non-enzymic (chemical) and enzymic attacks (Eriksson et al., 1990; Highley and Dashek, 1998). White-rot fungi have the capability to degrade lignin as well as other wood cell-wall components, although the rate at which they do so differs. Selective (or preferential) and simultaneous white-rots are categorized on the basis of removal of cell-wall constituents. Selective white-rots degrade hemicelluloses and lignin,
ARTICLE IN PRESS B. Mohebby / International Biodeterioration & Biodegradation 55 (2005) 247–251
resulting in defibrillation through dissolution of the middle lamella. In contrast, simultaneous or nonselective fungi remove lignin, hemicelluloses and cellulose at similar rates, resulting in homogeneous decay of the cell wall. The influence of white-rot decay on wood chemistry has been studied by various methods (Eriksson et al., 1990; Blanchette et al., 1990; Martinez et al., 2001). Soft-rot fungi remove all cell-wall constituents, resulting in collapse of cell walls. Type-I soft-rot fungi cause decay that is erosive and attacks all cell-wall layers simultaneously and removes all polymers at similar rates, while type-II selectively attack mostly polysaccharides in secondary wall layers, resulting in some developing typical cavities (Daniel and Nilsson, 1998). Chemistry of soft-rot fungi has been investigated by Levi and Peterson (1965), Savory and Pinion (1958) and Nilsson et al. (1989). All investigative methods mentioned above are tedious and time consuming, but FTIR spectroscopy provides a rapid way to qualitatively determine wood chemistry as well as recognizing decay (Pandey, 1999; Pandey and Theagarjan, 1997). Using this technique Pandey and Pitman (2003) were able to detect fungal decay in hard- and soft-wood. The reported method is convenient and rapid, but it requires preparation of small pellets with KBr. In the present study another method, attenuated total reflection (ATR) infrared spectroscopy, has been used as a very rapid and easier technique than FTIR to monitor chemical aspect of white-rot decay in wood. It is can be used for a few milligrams wood powder or solid samples without any previous preparation.
2. Material and methods Wood. Small beech (Fagus sylvatica) blocks ð5 5 20 mm3 Þ were prepared, dry weights of 10 mini-blocks were determined and autoclaved at 121 C for 20 min. Microorganism. The typical white-rot basidiomycete, Trametes versicolor ((L.: Fries) Pilat), obtained from the Forest Product Laboratory, Madison, Wisconsin, USA, was maintained at 4 C on slants peptone yeast extract agar of the composition ðg L1 Þ: glucose, 20; peptone, 5; yeast extract, 2; KH2 PO4 ; 1; MgSO4 5H2 O; 0.5; agar, 15. Culture. Glucose/malt extract plates containing (L1 ) 10 g glucose, 4.5 g malt extract and 15 g agar were prepared and autoclaved at 121 C for 20 min. Plates were inoculated with small pieces cut from stock cultures and the plates incubated at 27 C and 70% R.H. until the mycelium covered the plates (a few days). Thereafter, 3 mm were aseptically punched from the leading edge of the mycelium and transferred onto test plates. When mycelium had covered the test plates, oven-dried mini-blocks were placed directly on U-
shaped glass supports on the mycelium. The test plates were then incubated at 27 C and 70% R.H. for 84 days. At fortnightly intervals, 20 replicate mini-blocks were removed for analysis. Determination of mass loss. Samples were cleaned carefully and dried at 103 2 C to determine their dry mass after fungal attack. The mass losses of individual samples were calculated and used to determine mean percentage mass loss for each sampling time. IR spectroscopy. For ATR, dried samples were milled and passed through a mesh 40 sieve. IR spectra were obtained directly from wood powder on a detector prism. Using a Bruker Vectra 22 FTIR Spectrometer equipped with a DuraSampleIR IIt detector, all samples were examined at a spectral resolution of 4 cm1 : Spectra from 60 scans of sample and background scans were measured, the background spectra being collected using an empty collector. A rubber band method was used for baseline correction. The band for CO2 was removed to make a suitable baseline correction (Mohebby, 2003).
3. Results and discussion By 28 days, the mean mass loss for the wood blocks exposed to T. versicolor was 420% (Fig. 1) and by 70 and 84 days was approx. 51% and 60%, respectively. An IR spectrum of undecayed beech wood (Fig. 2) showed a strong OH bond vibration at 3500–3300 cm1 (1) relating to absorbed water in wood and a prominent C–H stretching vibration around 2881 cm1 (2). There are many well-defined peaks in the fingerprint region of 1800–600 cm1 : The assigned peaks in the region mentioned are unconjugated C QO stretching in xylan at 1724 cm1 (3); conjugated C–O stretching at 1587 cm1 (4); aromatic skeletal vibration at 1500 cm1 (5); C–H deformation in lignin and carbohydrates at wave numbers 1450 (6) and 1417 cm1 (7); C–H deformation in cellulose and hemicelluloses at 1363 cm1 (8); C–H vibration in cellulose and C1 –O vibration in syringyl derivatives at 1319 cm1 (9);
Mass loss (%)
248
100 90 80 70 60 50 40 30 20 10 0 0
14
28
42 56 Period (Days)
70
Fig. 1. Mass loss in white rot decayed beech wood.
84
ARTICLE IN PRESS B. Mohebby / International Biodeterioration & Biodegradation 55 (2005) 247–251
249
13
84 day 70 day 12
56 day 42 day
10
28 day
1 11 8 7 9 6
3 2
4
14 day 14
0 day
5
1800
1700
1600
1500
1400
1300
1200
1100
1000
900
800
Wave number (cm-1) 3600
3100
2600
2100
1600
1100
600
-1
Wave number (cm )
Fig. 3. IR spectra of decayed beech wood after various periods of white-rot attack.
Fig. 2. IR spectra of undecayed beech wood.
0 day
syringyl ring and C–O stretching in lignin and xylan at wave number 1226 cm1 (10); C–O–C vibration in cellulose and hemicellulose at 1151 cm1 (11); aromatic skeletal and C–O stretch at 1116 cm1 (12); C–O stretch in cellulose and hemicelluloses at wave number 1024 cm1 (13) and C–H deformation in cellulose at 892 cm1 (14). The IR spectra of white-rot decayed beech wood (Fig. 3) indicate changes of peaks in fingerprint regions at different stages of decay. Comparison between the peaks from different stages of attack with the undecayed wood reveals appearance of new peaks after 28 days of exposure to fungus, which are prominent indications of new bands obtained from altered cell wall constituents due to reaction with fungal enzymes. Subtraction of peaks obtained for attacked wood after 70 and 84 days from the undecayed beech clearly reveals the appearance of new peaks in the fingerprint region that are sometimes unknown (Figs. 4 and 5). In both Figs. 4 and 5, the baselines indicate no differences between the undecayed and the decayed wood; while the peaks under the baselines show increase of bands and above the baselines indicate prominent decrease of the bands. The spectra for wood exposed to the fungus for 70 days reveal appearance of new peaks around 1800–1650 cm1 and 1550–1500 cm1 ; the respective fingerprint regions for hemicelluloses (especially xylan) and aromatic derivatives of lignin. The increases of the peaks indicate that new bands occurred owing to breakdown of lignin and hemicellulose polymers. During fungal attack, different types of hydrolyzing enzyme could be liberated
1800
1700
1600
1500
1400
Subtracted for 70 days
1300
1200
1100
1000
900
800
-1
Wave number (cm ) Fig. 4. Subtracted IR spectra of white-rot decayed beech wood after 70 days.
to alter and break linkages in the cell-wall components and release them as small molecules. During this period, many chemical changes occurred in these constituents, indicating that the fungus could assimilate them as carbon sources. The lower peaks were removed and a wide range of peaks appeared above the baseline by 84 days (Fig. 5). The peaks above the baseline reveal removal of chemical constituents from the cell walls. The white-rot fungus could remove most of the hemicelluloses, lignin and some cellulose, according to the spectra and assigned
ARTICLE IN PRESS B. Mohebby / International Biodeterioration & Biodegradation 55 (2005) 247–251
250
0 Day
1800
1700
1600
1500
1400
Subtracted for 84 days
1300
1200
1100
1000
900
800
Wave number (cm-1) Fig. 5. Subtracted IR spectra of white-rot decayed beech wood after 84 days.
peaks (Table 1). The results confirmed that, having the ability to remove all types of the cell-wall constituents, T. versicolor is a non-selective white-rot fungus. Chemical analyses and microscopical findings in other studies have also shown that this species is a non-selective or simultaneous type and that it removes lignin, hemicelluloses and cellulose (Eriksson et al., 1990; Anagnost, 1998; Highley and Dashek, 1998; Mohebby, 2003). Pandey and Pitman (2003) studied chemical changes in beech wood decayed by T. versicolor by using FTIR spectroscopy. They showed decrease in intensity of bands for lignin and carbohydrate, particularly hemicelluloses, indicating that hemicelluloses are preferentially attacked by T. versicolor, rather than cellulose. Faix et al. (1993) studied chemical alteration in beech wood decayed by T. versicolor using FTIR and recorded complex bands between 1200 and 900 cm1 ; due mainly to polysaccharides, and hemicellulose bands at 1740 cm1 with small changes. However, decay results revealed increased intensity at 1646 cm1 band due to conjugated carbonyl groups originating from lignin, while the aromatic skeletal vibration band at 1596 cm1 decreased in intensity. The IR bands after 70 days of
Table 1 Assigned peaks of white rot decayed beech after 84 days of exposure Wave number
Assignment
Peak situationa
1724 1587 1500 1450
C QO unconjugated groups in lignin and carboxylic acid ester hemicellulosesb Conjugated C–Oc Aromatic skeletal; Benzene ring vibration in lignind C–H deformation in lignin and carbohydrates; CH3, CH2, benzene ring vibration in lignine C–H deformation in lignin and carbohydrates; Aromatic skeletal vibrations combined with C–H in plane deformationf C–H deformation in cellulose and hemicelluloses; C–H bending vibration in cellulose and hemicellulosesg C–H vibration in cellulose; C1 –O in syringyl derivativesh Syringyl ring; C–O stretch in lignin and xylani C–O–C vibration in cellulose and hemicellulosesj Aromatic skeletal; C–C stretch; O–H association band in cellulose and hemicellulosesk C–O stretch in cellulose and hemicelluloses; C–O of primary alcoholl C–H deformation in cellulose; C1 group frequency in cellulose and hemicellulosem
1417 1363 1319 1226 1151 1116 1024 892 a
Decreased band intensity; + increased band intensity. Takahashi et al. (1989). c Pandey and Pitman (2003). d Pandey and Theagarjan (1997); Schultz and Glasser (1986); Pandey (1999). e Pandey and Theagarjan (1997); Kimura et al. (1992). f Pandey and Theagarjan (1997); Pandey (1999); Rodrigues et al. (1998). g Evans et al. (1992); Pandey and Theagarjan (1997); Pandey and Pitman (2003). h Pandey and Theagarjan (1997); Pandey (1999); Pandey and Pitman (2003). i Pandey and Pitman (2003); Faix (1991). j Pandey and Theagarjan (1997); Rodrigues et al. (1998). k Schultz and Glasser (1986); Pandey (1999). l Pandey (1999); Pandey and Theagarjan (1997). m Pandey and Theagarjan (1997); Rodrigues et al. (1998). b
+
ARTICLE IN PRESS B. Mohebby / International Biodeterioration & Biodegradation 55 (2005) 247–251
exposure in the present study compare with the results of Faix et al. (1993) in pointing to sequential decay of the cell-wall constituents lignin, hemicellulose and then cellulose. In summary, the ATR spectroscopy technique used here to examine qualitatively chemical alterations in beech wood decayed by T. versicolor has shown that this fungus decays the cell-wall components non-selectively and removes lignin, hemicelluloses and, partially, cellulose, agreeing with the work of previous authors who have used IR spectroscopy. ATR spectroscopy has proved to be convenient and applicable technique that gives rapid results from decayed wood. References Anagnost, S.E., 1998. Light microscopic diagnosis of wood decay. IAWA Journal 19, 141–167. Bjurman, J., Wadso¨, L., 2000. Microcalorimetric measurements of metabolic activity of six decay fungi on spruce wood as a function of temperature. Mycologia 92, 23–28. Blanchette, R.A., Nilsson, T., Daniel, G., Abad, A., 1990. Biological degradation of wood. In: Rowell, R.M., Barbour, R.J. (Eds.), Archaeological Wood. Advances in Chemistry Series, No. 225. American Chemical Society, Washington, DC, pp. 141–192. Clausen, C., 1996. Immunological detection of wood decay fungi, an overview of techniques developed from 1986 to the present. International Biodeterioration and Biodegradation 39, 133–143. Clausen, C., Green, F., 1998. Immunodiagnostic wood decay test. Techline: decay process and bioprocessing, USDA Forest Products Laboratory, II-3, online at http://www.fpl.fs.fed.us. Daniel, G., Nilsson, T., 1998. Development in the study of soft rot and bacterial decay. In: Bruce, A., Palfreyman, J.W. (Eds.), Forest Products Biotechnology. Taylor and Francis, London, pp. 37–62. Eriksson, K.E.L., Blanchette, R.A., Ander, P., 1990. Microbial and Enzymatic Degradation of Wood and Wood Components. Springer, Berlin. Evans, P.D., Micehll, A.J., Schmalzl, K.J., 1992. Studies of the degradation and protection of wood surfaces. Wood Science and Technology 26, 151–163. Faix, O., 1991. Classification of lignins from different botanical origins by FT-IR spectroscopy. Holzforschung 45 (Suppl.), 21–27. Faix, O., Bremer, J., Schmidtt, O., Stevanovic, T., 1993. Monitoring of chemical changes in white-rot degraded beech wood by pyrolysisgas chromatography and Fourier transform infrared spectroscopy. Journal of Analytical and Applied Pyrolysis 21, 147–162. Highley, T.L., Dashek, W.V., 1998. Biotechnology in the study of brown- and white-rot decay. In: Bruce, A., Palfreyman, J.W. (Eds.), Forest Products Biotechnology. Taylor and Francis, London, pp. 15–36. Illman, B.L., Bajt, S., 1997. Nondestructive elemental analysis of wood biodeterioration using electron paramagnetic resonance and synchrotron X-ray fluorescence. International Biodeterioration and Biodegradation 39, 235–243. Kimura, F., Kimura, T., Gray, D.G., 1992. FT-IR study of UVirradiated stoneground wood pulp. Holzforschung 46, 529–532. Levi, M.P., Peterson, R.D., 1965. A chemical and microscopic examination of the action of the soft rot fungus Chaetomium globosum on beech wood (Fagus slv.). Holzforschung 19, 183–190. Machek, L., Edlund, M.-L., Sierra-Alvarez, R., Militz, H., 1998a. Acoustic technique for assessing decay in preservative treated wood. IRG Document No. IRG/WP 98-20138. The International Research Group on Wood Preservation, Stockholm.
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Machek, L., Militz, H., Sierra-Alvarez, R., 1998b. A dynamic approach to assess the modulus of elasticity in wood decay testing. IRG Document No. IRG/WP 98-20139. The International Research Group on Wood Preservation, Stockholm. Martinez, A.T., Camarero, S., Gutie´rrez, A., Bochini, P., Galleti, G.C., 2001. Studies on wheat lignin degradation by Pleurotus species using analytical pyrolysis. Journal of Analytical and Applied Pyrolysis 58–59, 401–411. Mohebby, B., 2003. Biological attack of acetylated wood. Ph.D. Thesis, Go¨ttingen University, Go¨ttingen, Germany. Mohebby, B., Militz, H., 2002. Soft rot decay in acetylated wood. IRG Document No. IRG/WP 02-40231. The International Research Group on Wood Preservation, Stockholm. Mohebby, B., Mai, C., Militz, H., 2003. Soft rot decay in acetylated wood: microcalorimetry and ergosterol assay in decayed wood. Proceedings, European Conference on Wood Modification, Ghent, Belgium, 3–4 April 2003, pp. 197–202. Nilsson, K., 1998. Detection of wood decay using electronic nose. Ph.D. Thesis, Swedish University of Agricultural Sciences, Uppsala, Sweden, 50. Nilsson, T., Daniel, G., Kirk, T.K., Obst, J.R., 1989. Chemistry and microscopy of wood decay by some higher ascomycetes. Holzforschung 43, 11–18. Ouis, D., 2000. Detection of decay in logs through measuring the damping of bending vibrations by means of a room acoustical technique. Wood Science and Technology 34, 221–236. Pandey, K.K., 1999. A study of chemical structure of soft and hardwood polymers by FTIR spectroscopy. Journal of Applied Polymer Sciences 71, 1969–1975. Pandey, K.K., Pitman, A.J., 2003. FTIR studies of the changes in wood following decay by brown-rot and white-rot fungi. International Biodeterioration and Biodegradation 52, 151–160. Pandey, K.K., Theagarjan, K.S., 1997. Analysis of wood surface and ground wood by diffuse reflectance (DRIFT) and photoacoustic (PAS) Fourier transform infra red spectroscopy. Holz als Roh- und Werkstoff 55, 383–390. Rinn, F., Schweingruber, F.H., Scha¨r, E., 1996. Resistograph and X-ray density charts of wood comparative evaluation of drill resistance profiles and X-ray density charts of different wood species. Holzforschung 50, 303–311. Rodrigues, J., Faix, O., Pereira, H., 1998. Determination of lignin content of Eucalyptus globulus wood using FTIR spectroscopy. Holzforschung 52, 46–50. Ross, R.J., de Groot, R.C., 1998. Scanning technique to identify biological degraded wood. Experimental Techniques, May/June, 32–33. Savory, J.G., Pinion, L.C., 1958. Chemical aspects of decay of beech wood by Chaetomium globosum. Holzforschung 12, 99–103. Schultz, T.P., Glasser, W.G., 1986. Quantitative structural analysis of lignin by diffuse reflectance Fourier transform infrared spectrometry. Holzforschung 40 (Suppl.), 37–44. Takahashi, M., Imamura, Y., Tanahashi, M., 1989. Effect of acetylation on decay resistance of wood against brown rot, white rot and soft rot. International Chemistry, Congress of Pacific Basin Societies, Agrochemistry, Sub-symposium on Chemical Modification of lignocellulosic Materials—Chemical Reactions, p. 16. Wa¨lchli, O., 1970. Influence of the content of organic matter of soil on the degradation of wood by soft rot fungi. IRG Document No. IRG/WP 27. The International Research Group on Wood Preservation, Stockholm. Xie, Y., Bjurman, J., Wadso¨, L., 1997. Microcalorimetric characterization of the recovery of brown rot fungus after exposure to high and low temperature, oxygen depletion and drying. Holzforschung 51, 201–206. Zabell, R.A., Morrell, J.J., 1992. Wood Microbiology, Decay and its Prevention. Academic Press, New York.
International Biodeterioration & Biodegradation 55 (2005) 247–251 www.elsevier.com/locate/ibiod
Attenuated total reflection infrared spectroscopy of white-rot decayed beech wood Behbood Mohebby Department of Wood and Paper Sciences, Faculty of Natural Resources and Marine Sciences, Tarbiat Modarress University, P.O. Box 46414-356, Noor, Iran Received 4 August 2004; received in revised form 13 November 2004; accepted 29 December 2004
Abstract Small blocks of beech wood were exposed to the white-rot fungus Trametes versicolor for a period of 84 days to investigate chemical alteration in decayed wood by infrared spectroscopy. Decayed samples were analyzed at 2 week intervals by using attenuated total teflection (ATR) infrared spectroscopy as a rapid method. Analyses showed that chemical alteration in wood began after the second week of exposure. The appearance of new peaks indicated chemical modification of cell walls between days 28 and 70 of exposure to the fungus, and the disappearance of the peaks at day 84 indicates removal of the cell wall constituents. This investigation showed that ATR spectroscopy is a very applicable and rapid method for studying wood biodegradation. r 2005 Elsevier Ltd. All rights reserved. Keywords: Attenuated total reflection infrared spectroscopy; Beech; White-rot decay; Trametes versicolor
1. Introduction Several types of biodegradation have been recognized in wood, viz. fungal decay, bacterial degradation and insect attack. Fungal decay is the most important and widespread type of degradation and is caused by white-, brown- or soft-rot fungi (Zabell and Morrell, 1992; Eriksson et al., 1990). Different methods and techniques have been applied to monitor fungal decay, including gravimetric analysis (see, for example, Wa¨lchli, 1970); loss of dynamic modulus of elasticity (Machek et al., 1998a, b; Mohebby and Militz, 2002; Mohebby, 2003); technical microscopy, e.g. light, polarized and electron microscopies (Anagnost, 1998); NMR and FTIR spectroscopy (Pandey and Pitman, 2003); microcalorimetry (Xie et al., 1997; Bjurman and Wadso¨, 2000; Mohebby, 2003; Mohebby et al., 2003), ergosterol assay (Mohebby, 2003; Mohebby et al., 2003); analysis of flourescein diacetate (FDA) (Mohebby, 2003); GC–MS spectroscopy; chemical analysis (Zabell and Morrell, E-mail address: [email protected]. 0964-8305/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2005.01.003
1992; Mohebby and Militz, 2002); electronic nose (Nilsson, 1998); electron paramagnetic resonance and synchrotron X-ray fluorescence (Illman and Bajt, 1997); immunodiagnostics (Clausen, 1996; Clausen and Green, 1998); bending vibration technique (Ouis, 2000); resistography (Rinn et al., 1996); and scanning technique (Ross and de Groot, 1998). White-, brown- and soft-rot fungi attack wood based on their enzymatic systems; these enzymes penetrate the wood cell wall, alter its chemistry and break down the cell-wall polymers into constituents that can be taken up by hyphae. Brown-rot fungi selectively decay cell-wall polysaccharides, with limited lignin degradation. The decay system in this type is based on both non-enzymic (chemical) and enzymic attacks (Eriksson et al., 1990; Highley and Dashek, 1998). White-rot fungi have the capability to degrade lignin as well as other wood cell-wall components, although the rate at which they do so differs. Selective (or preferential) and simultaneous white-rots are categorized on the basis of removal of cell-wall constituents. Selective white-rots degrade hemicelluloses and lignin,
ARTICLE IN PRESS B. Mohebby / International Biodeterioration & Biodegradation 55 (2005) 247–251
resulting in defibrillation through dissolution of the middle lamella. In contrast, simultaneous or nonselective fungi remove lignin, hemicelluloses and cellulose at similar rates, resulting in homogeneous decay of the cell wall. The influence of white-rot decay on wood chemistry has been studied by various methods (Eriksson et al., 1990; Blanchette et al., 1990; Martinez et al., 2001). Soft-rot fungi remove all cell-wall constituents, resulting in collapse of cell walls. Type-I soft-rot fungi cause decay that is erosive and attacks all cell-wall layers simultaneously and removes all polymers at similar rates, while type-II selectively attack mostly polysaccharides in secondary wall layers, resulting in some developing typical cavities (Daniel and Nilsson, 1998). Chemistry of soft-rot fungi has been investigated by Levi and Peterson (1965), Savory and Pinion (1958) and Nilsson et al. (1989). All investigative methods mentioned above are tedious and time consuming, but FTIR spectroscopy provides a rapid way to qualitatively determine wood chemistry as well as recognizing decay (Pandey, 1999; Pandey and Theagarjan, 1997). Using this technique Pandey and Pitman (2003) were able to detect fungal decay in hard- and soft-wood. The reported method is convenient and rapid, but it requires preparation of small pellets with KBr. In the present study another method, attenuated total reflection (ATR) infrared spectroscopy, has been used as a very rapid and easier technique than FTIR to monitor chemical aspect of white-rot decay in wood. It is can be used for a few milligrams wood powder or solid samples without any previous preparation.
2. Material and methods Wood. Small beech (Fagus sylvatica) blocks ð5 5 20 mm3 Þ were prepared, dry weights of 10 mini-blocks were determined and autoclaved at 121 C for 20 min. Microorganism. The typical white-rot basidiomycete, Trametes versicolor ((L.: Fries) Pilat), obtained from the Forest Product Laboratory, Madison, Wisconsin, USA, was maintained at 4 C on slants peptone yeast extract agar of the composition ðg L1 Þ: glucose, 20; peptone, 5; yeast extract, 2; KH2 PO4 ; 1; MgSO4 5H2 O; 0.5; agar, 15. Culture. Glucose/malt extract plates containing (L1 ) 10 g glucose, 4.5 g malt extract and 15 g agar were prepared and autoclaved at 121 C for 20 min. Plates were inoculated with small pieces cut from stock cultures and the plates incubated at 27 C and 70% R.H. until the mycelium covered the plates (a few days). Thereafter, 3 mm were aseptically punched from the leading edge of the mycelium and transferred onto test plates. When mycelium had covered the test plates, oven-dried mini-blocks were placed directly on U-
shaped glass supports on the mycelium. The test plates were then incubated at 27 C and 70% R.H. for 84 days. At fortnightly intervals, 20 replicate mini-blocks were removed for analysis. Determination of mass loss. Samples were cleaned carefully and dried at 103 2 C to determine their dry mass after fungal attack. The mass losses of individual samples were calculated and used to determine mean percentage mass loss for each sampling time. IR spectroscopy. For ATR, dried samples were milled and passed through a mesh 40 sieve. IR spectra were obtained directly from wood powder on a detector prism. Using a Bruker Vectra 22 FTIR Spectrometer equipped with a DuraSampleIR IIt detector, all samples were examined at a spectral resolution of 4 cm1 : Spectra from 60 scans of sample and background scans were measured, the background spectra being collected using an empty collector. A rubber band method was used for baseline correction. The band for CO2 was removed to make a suitable baseline correction (Mohebby, 2003).
3. Results and discussion By 28 days, the mean mass loss for the wood blocks exposed to T. versicolor was 420% (Fig. 1) and by 70 and 84 days was approx. 51% and 60%, respectively. An IR spectrum of undecayed beech wood (Fig. 2) showed a strong OH bond vibration at 3500–3300 cm1 (1) relating to absorbed water in wood and a prominent C–H stretching vibration around 2881 cm1 (2). There are many well-defined peaks in the fingerprint region of 1800–600 cm1 : The assigned peaks in the region mentioned are unconjugated C QO stretching in xylan at 1724 cm1 (3); conjugated C–O stretching at 1587 cm1 (4); aromatic skeletal vibration at 1500 cm1 (5); C–H deformation in lignin and carbohydrates at wave numbers 1450 (6) and 1417 cm1 (7); C–H deformation in cellulose and hemicelluloses at 1363 cm1 (8); C–H vibration in cellulose and C1 –O vibration in syringyl derivatives at 1319 cm1 (9);
Mass loss (%)
248
100 90 80 70 60 50 40 30 20 10 0 0
14
28
42 56 Period (Days)
70
Fig. 1. Mass loss in white rot decayed beech wood.
84
ARTICLE IN PRESS B. Mohebby / International Biodeterioration & Biodegradation 55 (2005) 247–251
249
13
84 day 70 day 12
56 day 42 day
10
28 day
1 11 8 7 9 6
3 2
4
14 day 14
0 day
5
1800
1700
1600
1500
1400
1300
1200
1100
1000
900
800
Wave number (cm-1) 3600
3100
2600
2100
1600
1100
600
-1
Wave number (cm )
Fig. 3. IR spectra of decayed beech wood after various periods of white-rot attack.
Fig. 2. IR spectra of undecayed beech wood.
0 day
syringyl ring and C–O stretching in lignin and xylan at wave number 1226 cm1 (10); C–O–C vibration in cellulose and hemicellulose at 1151 cm1 (11); aromatic skeletal and C–O stretch at 1116 cm1 (12); C–O stretch in cellulose and hemicelluloses at wave number 1024 cm1 (13) and C–H deformation in cellulose at 892 cm1 (14). The IR spectra of white-rot decayed beech wood (Fig. 3) indicate changes of peaks in fingerprint regions at different stages of decay. Comparison between the peaks from different stages of attack with the undecayed wood reveals appearance of new peaks after 28 days of exposure to fungus, which are prominent indications of new bands obtained from altered cell wall constituents due to reaction with fungal enzymes. Subtraction of peaks obtained for attacked wood after 70 and 84 days from the undecayed beech clearly reveals the appearance of new peaks in the fingerprint region that are sometimes unknown (Figs. 4 and 5). In both Figs. 4 and 5, the baselines indicate no differences between the undecayed and the decayed wood; while the peaks under the baselines show increase of bands and above the baselines indicate prominent decrease of the bands. The spectra for wood exposed to the fungus for 70 days reveal appearance of new peaks around 1800–1650 cm1 and 1550–1500 cm1 ; the respective fingerprint regions for hemicelluloses (especially xylan) and aromatic derivatives of lignin. The increases of the peaks indicate that new bands occurred owing to breakdown of lignin and hemicellulose polymers. During fungal attack, different types of hydrolyzing enzyme could be liberated
1800
1700
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Subtracted for 70 days
1300
1200
1100
1000
900
800
-1
Wave number (cm ) Fig. 4. Subtracted IR spectra of white-rot decayed beech wood after 70 days.
to alter and break linkages in the cell-wall components and release them as small molecules. During this period, many chemical changes occurred in these constituents, indicating that the fungus could assimilate them as carbon sources. The lower peaks were removed and a wide range of peaks appeared above the baseline by 84 days (Fig. 5). The peaks above the baseline reveal removal of chemical constituents from the cell walls. The white-rot fungus could remove most of the hemicelluloses, lignin and some cellulose, according to the spectra and assigned
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1800
1700
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Subtracted for 84 days
1300
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Wave number (cm-1) Fig. 5. Subtracted IR spectra of white-rot decayed beech wood after 84 days.
peaks (Table 1). The results confirmed that, having the ability to remove all types of the cell-wall constituents, T. versicolor is a non-selective white-rot fungus. Chemical analyses and microscopical findings in other studies have also shown that this species is a non-selective or simultaneous type and that it removes lignin, hemicelluloses and cellulose (Eriksson et al., 1990; Anagnost, 1998; Highley and Dashek, 1998; Mohebby, 2003). Pandey and Pitman (2003) studied chemical changes in beech wood decayed by T. versicolor by using FTIR spectroscopy. They showed decrease in intensity of bands for lignin and carbohydrate, particularly hemicelluloses, indicating that hemicelluloses are preferentially attacked by T. versicolor, rather than cellulose. Faix et al. (1993) studied chemical alteration in beech wood decayed by T. versicolor using FTIR and recorded complex bands between 1200 and 900 cm1 ; due mainly to polysaccharides, and hemicellulose bands at 1740 cm1 with small changes. However, decay results revealed increased intensity at 1646 cm1 band due to conjugated carbonyl groups originating from lignin, while the aromatic skeletal vibration band at 1596 cm1 decreased in intensity. The IR bands after 70 days of
Table 1 Assigned peaks of white rot decayed beech after 84 days of exposure Wave number
Assignment
Peak situationa
1724 1587 1500 1450
C QO unconjugated groups in lignin and carboxylic acid ester hemicellulosesb Conjugated C–Oc Aromatic skeletal; Benzene ring vibration in lignind C–H deformation in lignin and carbohydrates; CH3, CH2, benzene ring vibration in lignine C–H deformation in lignin and carbohydrates; Aromatic skeletal vibrations combined with C–H in plane deformationf C–H deformation in cellulose and hemicelluloses; C–H bending vibration in cellulose and hemicellulosesg C–H vibration in cellulose; C1 –O in syringyl derivativesh Syringyl ring; C–O stretch in lignin and xylani C–O–C vibration in cellulose and hemicellulosesj Aromatic skeletal; C–C stretch; O–H association band in cellulose and hemicellulosesk C–O stretch in cellulose and hemicelluloses; C–O of primary alcoholl C–H deformation in cellulose; C1 group frequency in cellulose and hemicellulosem
1417 1363 1319 1226 1151 1116 1024 892 a
Decreased band intensity; + increased band intensity. Takahashi et al. (1989). c Pandey and Pitman (2003). d Pandey and Theagarjan (1997); Schultz and Glasser (1986); Pandey (1999). e Pandey and Theagarjan (1997); Kimura et al. (1992). f Pandey and Theagarjan (1997); Pandey (1999); Rodrigues et al. (1998). g Evans et al. (1992); Pandey and Theagarjan (1997); Pandey and Pitman (2003). h Pandey and Theagarjan (1997); Pandey (1999); Pandey and Pitman (2003). i Pandey and Pitman (2003); Faix (1991). j Pandey and Theagarjan (1997); Rodrigues et al. (1998). k Schultz and Glasser (1986); Pandey (1999). l Pandey (1999); Pandey and Theagarjan (1997). m Pandey and Theagarjan (1997); Rodrigues et al. (1998). b
+
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exposure in the present study compare with the results of Faix et al. (1993) in pointing to sequential decay of the cell-wall constituents lignin, hemicellulose and then cellulose. In summary, the ATR spectroscopy technique used here to examine qualitatively chemical alterations in beech wood decayed by T. versicolor has shown that this fungus decays the cell-wall components non-selectively and removes lignin, hemicelluloses and, partially, cellulose, agreeing with the work of previous authors who have used IR spectroscopy. ATR spectroscopy has proved to be convenient and applicable technique that gives rapid results from decayed wood. References Anagnost, S.E., 1998. Light microscopic diagnosis of wood decay. IAWA Journal 19, 141–167. Bjurman, J., Wadso¨, L., 2000. Microcalorimetric measurements of metabolic activity of six decay fungi on spruce wood as a function of temperature. Mycologia 92, 23–28. Blanchette, R.A., Nilsson, T., Daniel, G., Abad, A., 1990. Biological degradation of wood. In: Rowell, R.M., Barbour, R.J. (Eds.), Archaeological Wood. Advances in Chemistry Series, No. 225. American Chemical Society, Washington, DC, pp. 141–192. Clausen, C., 1996. Immunological detection of wood decay fungi, an overview of techniques developed from 1986 to the present. International Biodeterioration and Biodegradation 39, 133–143. Clausen, C., Green, F., 1998. Immunodiagnostic wood decay test. Techline: decay process and bioprocessing, USDA Forest Products Laboratory, II-3, online at http://www.fpl.fs.fed.us. Daniel, G., Nilsson, T., 1998. Development in the study of soft rot and bacterial decay. In: Bruce, A., Palfreyman, J.W. (Eds.), Forest Products Biotechnology. Taylor and Francis, London, pp. 37–62. Eriksson, K.E.L., Blanchette, R.A., Ander, P., 1990. Microbial and Enzymatic Degradation of Wood and Wood Components. Springer, Berlin. Evans, P.D., Micehll, A.J., Schmalzl, K.J., 1992. Studies of the degradation and protection of wood surfaces. Wood Science and Technology 26, 151–163. Faix, O., 1991. Classification of lignins from different botanical origins by FT-IR spectroscopy. Holzforschung 45 (Suppl.), 21–27. Faix, O., Bremer, J., Schmidtt, O., Stevanovic, T., 1993. Monitoring of chemical changes in white-rot degraded beech wood by pyrolysisgas chromatography and Fourier transform infrared spectroscopy. Journal of Analytical and Applied Pyrolysis 21, 147–162. Highley, T.L., Dashek, W.V., 1998. Biotechnology in the study of brown- and white-rot decay. In: Bruce, A., Palfreyman, J.W. (Eds.), Forest Products Biotechnology. Taylor and Francis, London, pp. 15–36. Illman, B.L., Bajt, S., 1997. Nondestructive elemental analysis of wood biodeterioration using electron paramagnetic resonance and synchrotron X-ray fluorescence. International Biodeterioration and Biodegradation 39, 235–243. Kimura, F., Kimura, T., Gray, D.G., 1992. FT-IR study of UVirradiated stoneground wood pulp. Holzforschung 46, 529–532. Levi, M.P., Peterson, R.D., 1965. A chemical and microscopic examination of the action of the soft rot fungus Chaetomium globosum on beech wood (Fagus slv.). Holzforschung 19, 183–190. Machek, L., Edlund, M.-L., Sierra-Alvarez, R., Militz, H., 1998a. Acoustic technique for assessing decay in preservative treated wood. IRG Document No. IRG/WP 98-20138. The International Research Group on Wood Preservation, Stockholm.
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Machek, L., Militz, H., Sierra-Alvarez, R., 1998b. A dynamic approach to assess the modulus of elasticity in wood decay testing. IRG Document No. IRG/WP 98-20139. The International Research Group on Wood Preservation, Stockholm. Martinez, A.T., Camarero, S., Gutie´rrez, A., Bochini, P., Galleti, G.C., 2001. Studies on wheat lignin degradation by Pleurotus species using analytical pyrolysis. Journal of Analytical and Applied Pyrolysis 58–59, 401–411. Mohebby, B., 2003. Biological attack of acetylated wood. Ph.D. Thesis, Go¨ttingen University, Go¨ttingen, Germany. Mohebby, B., Militz, H., 2002. Soft rot decay in acetylated wood. IRG Document No. IRG/WP 02-40231. The International Research Group on Wood Preservation, Stockholm. Mohebby, B., Mai, C., Militz, H., 2003. Soft rot decay in acetylated wood: microcalorimetry and ergosterol assay in decayed wood. Proceedings, European Conference on Wood Modification, Ghent, Belgium, 3–4 April 2003, pp. 197–202. Nilsson, K., 1998. Detection of wood decay using electronic nose. Ph.D. Thesis, Swedish University of Agricultural Sciences, Uppsala, Sweden, 50. Nilsson, T., Daniel, G., Kirk, T.K., Obst, J.R., 1989. Chemistry and microscopy of wood decay by some higher ascomycetes. Holzforschung 43, 11–18. Ouis, D., 2000. Detection of decay in logs through measuring the damping of bending vibrations by means of a room acoustical technique. Wood Science and Technology 34, 221–236. Pandey, K.K., 1999. A study of chemical structure of soft and hardwood polymers by FTIR spectroscopy. Journal of Applied Polymer Sciences 71, 1969–1975. Pandey, K.K., Pitman, A.J., 2003. FTIR studies of the changes in wood following decay by brown-rot and white-rot fungi. International Biodeterioration and Biodegradation 52, 151–160. Pandey, K.K., Theagarjan, K.S., 1997. Analysis of wood surface and ground wood by diffuse reflectance (DRIFT) and photoacoustic (PAS) Fourier transform infra red spectroscopy. Holz als Roh- und Werkstoff 55, 383–390. Rinn, F., Schweingruber, F.H., Scha¨r, E., 1996. Resistograph and X-ray density charts of wood comparative evaluation of drill resistance profiles and X-ray density charts of different wood species. Holzforschung 50, 303–311. Rodrigues, J., Faix, O., Pereira, H., 1998. Determination of lignin content of Eucalyptus globulus wood using FTIR spectroscopy. Holzforschung 52, 46–50. Ross, R.J., de Groot, R.C., 1998. Scanning technique to identify biological degraded wood. Experimental Techniques, May/June, 32–33. Savory, J.G., Pinion, L.C., 1958. Chemical aspects of decay of beech wood by Chaetomium globosum. Holzforschung 12, 99–103. Schultz, T.P., Glasser, W.G., 1986. Quantitative structural analysis of lignin by diffuse reflectance Fourier transform infrared spectrometry. Holzforschung 40 (Suppl.), 37–44. Takahashi, M., Imamura, Y., Tanahashi, M., 1989. Effect of acetylation on decay resistance of wood against brown rot, white rot and soft rot. International Chemistry, Congress of Pacific Basin Societies, Agrochemistry, Sub-symposium on Chemical Modification of lignocellulosic Materials—Chemical Reactions, p. 16. Wa¨lchli, O., 1970. Influence of the content of organic matter of soil on the degradation of wood by soft rot fungi. IRG Document No. IRG/WP 27. The International Research Group on Wood Preservation, Stockholm. Xie, Y., Bjurman, J., Wadso¨, L., 1997. Microcalorimetric characterization of the recovery of brown rot fungus after exposure to high and low temperature, oxygen depletion and drying. Holzforschung 51, 201–206. Zabell, R.A., Morrell, J.J., 1992. Wood Microbiology, Decay and its Prevention. Academic Press, New York.