SELECTED PROPERTIES OF BEECH WOOD BIODEGRADED BY WHITE-ROT FUNGI STANISLAV KURJATKO, RASTISLAV SOLÁR, MIROSLAVA MAMOŇOVÁ & JOZEF HUDEC Technical university in Zvolen, T. G. Masaryka 24, 960 01 Zvolen, Slovakia
[email protected]
ABSTRACT Presented paper deals with the influence of biotic degradation on the permeability and surface swelling of normal and tension beech in polar solvents. Biodegradations were performed by erosive strain of Phanerochaete chrysosporium and lignin selective strain Ceriporiopsis subvermispora. A 30-day biodegradation by erosive fungus resulted in a low weight loss of the normal wood (Tab. 1) and in increase of its permeability (Tab. 2a). Similar effects have been observed also for tension beech wood degraded by the same fungus. On the other hand, the application of lignin-selective fungus significantly reduced permeability of the both normal and tension wood (Tab. 2b). SEM study of split surfaces of sound and biodegraded samples of wood revealed significant alterations in micro-structure of biodegraded materials (Figs. 1a–c, 2a–c, 3a–c and 4a–c). Biodegradation of selected samples of beech wood extremely increased the kinetics of their surface swelling in plane perpendicular to grain. Structural changes of normal and tension beech wood due to biodegradation, reflected in its altered permeability and intensity of interactions with polar liquids, may positively influence penetration of air-dry wood with pulping liquors, protective agents and solutions of monomers or macromolecular compounds. Keywords: tension and normal beech wood, biodegradation, permeability, SEM, swelling
INTRODUCTION The action of white-rot fungi in the most of cases starts from lumina towards middle lamelae. As soon as the process of biodegradation starts the fast delignification of secondary walls is proceeding (MESSNER & SREBOTNIK 1994). The initial phases of the white-rot are accompanied by negligible degradation of the cell walls, however marked is the increased swelling of their released structure (NEČESANÝ 1963, MESSNER & SREBOTNIK 1994). This phenomenon (biopulping effect) together with a partial delignification are the main cause of reduced physical properties and mechanical strength of biodegraded wood. In proceeding degradation a release of the fibrilar cell wall structure, together with eroded areas and holes, mostly allocated in the pits become apparent. A separation of the cell walls layers, and delignification of the middle lamellae in later stages of decay have also been reported (LIESE & SCHMID 1966, MESSNER & SREBOTNIK 1994, SOLAR et al. 2001). According to BLANCHETTE (1988), wood parenchyma is sensitive to fungal attack, fibers undergo to decay more slowly, and the most resistant elements are the vessels, considered by the author for almost indegradable. Late stages of wood degradation by non-selective white-rot fungi are characterized by increase in the number and dimensions of erosion holes, released cellulose fibers, and by reduced width of the cell walls residua Wood Structure and Properties ´02 edited by J. Kúdela & S. Kurjatko, pp. 57–62 2002 Arbora Publishers, Zvolen, Slovakia ISBN 80–967088–9–9
(NEČESANÝ 1963, SCHMID & LIESE 1964). The effect of lignin selective fungi is less pronounced in respect to erosions, and fibres degradation, apparent however is the S2 layers delignification (ERIKSSON 1988). From the viewpoint of potential application of white-rot fungi for pretreatment of raw materials prior to pulping a medium-term bio-degradation seems to be the most convenient. This results in reduced lignin content, diminished mechanical strength and acceptable weight loss of wood. The aim of this contribution was to examine the alterations of beech wood structure, permeability and swelling resulting from its medium-term biodegradation with erosive (Phanerochaete chrysosporium K-3) and lignin-selective (Ceriporiopsis subvermispora CBS-374.636) strains of white-rot fungi. The above mentioned physical properties play an important part in wood penetration, and may affect the course of semichemical and chemical pulping processes significantly. The alterations of tension wood following from fungal biodegradation have been also investigated due to its apparently different structure and physico-mechanical properties.
MATERIALS AND METHODS The specimens of normal and tension beech wood for determination of permeability and kinetics of surface swelling have been selected from the sapwood of two 57
80 years old trunks. Dimensions of the specimens, sterilisation and conditions of biodegradations were described in detail in (SOLÁR et al. 2002). Coefficients of permeability (in direction parallel to grain) of sound and of the same biodegraded specimens prior and after saturation with ethanol/water mixture (1:1 v/v) were determined by the method of REGINÁČ et al. (1977). Kinetics of the specimens facial surface swelling was monitored by Meopta DN 45 microscope equipped by a CDD 100 E video Mitsubishi camera with PC processing the data by LUCIA software. The initial moisture content of sound and biodegraded normal wood was 2.5 %, in case of tension wood it equaled 2.8 %. Microscopy of split surfaces of the samples were performed using scanning electron microscope Tesla BS-300.
to deeper degradation of beech wood, tension wood being more susceptible to biotic attack. In Tab. 2 the coefficients of permeability in direction along the fibers are presented.
Figure 1a Vessel of sound normal spring wood, intact pits membranes issuing to ray parenchyma.
RESULTS AND DISCUSSION Data in Table 1 are concerning the weight loss of normal and of tension beech wood resulting from 30 days action of the applied fungi. Table 1 Weight loss of normal and tension beech wood due to 30-day fungal degradation Sample
Fungus
Normal wood Tension wood
Phanerochaete chrysosporium Ceriporiopsis subvermispora Phanerochaete chrysosporium Ceriporiopsis subvermispora
Weight loss [%] 6.24 3.01 10.10 5.97
As seen from Tab. 1, the weight loss of a substrate depended on the both biotic agent and sample of wood degraded. Erosive strain of white-rot fungus led
Figure 2a Vessel of sound tension spring wood, intact pit membranes issuing into ray parenchyma.
Table 2 Coefficients of permeability of sound and 30 days biodegraded normal (a) and tension (b) beech wood at the moisture contents of 15 % and above FSP (medium - ethanol/water 1:1 v/v) a) normal wood Degraded by Degraded by Phanerochaete Ceriporiopsis Sound wood Sound wood chrysosporium subvermispora Property w = 15 % w = 15 % w = 15 % w › BNV w = 15 % w › BNV x (m2) Sr (%) n b) tension wood
1.391011 36.64 28
Sound wood Property
x (m2) Sr (%) n
58
1.731011 32.96 28
1.711011 33.92 28
Degraded by Phanerochaete chrysosporium
1.401011 36.23 28
Sound wood
5.821012 47.03 28
6.151012 44.97 28
Degraded by Ceriporiopsis subvermispora
w = 15 %
w = 15 %
w › BNV
w = 15 %
w = 15 %
w › BNV
6.111012 48.63 27
1.9611011 50.67 27
1.0511011 43.16 27
5.541012 50.34 29
2.961012 45.91 29
3.521012 47.39 29
Wood structure and Properties ´02
Figure 1b Normal spring wood vessels, pattern od middle lamellae delignification, enzymatic dissolution of pits membranes between vessels.(Phanerochaete chrysosporium)
Figure 2b Enzymatic dissolution of pits membranes in the vessel of tension spring wood. Delignification of middle lamela and ruptures formation in the secondary wall of the vessel. (Phanerochaete chrysosporium)
Figure 1c Vessels of normal spring wood, enzymatically dissolved pits membrnes. Cut fibre – resistant cell wals, degradation only in the middle lamella. (Ceriporiopsis subvermispora)
Figure 2c Hyphae in tension spring wood, pit membranes relatively resistant. (Ceriporiopsis subvermispora)
Figure 3a Sound normal late wood, pattern of moderate ladder-like perforation in the vessel
Figure 4a Sound tension wood, incomplete ladder-like perforations of late wood vessels
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Figure 3b Normal late wood, absence of the warty layer of secondary wall in the vessel, hyphae growing through the ladder-like perforation and dissolving enzymatically the pits membranes (Phanerochaete chrysosporium)
Figure 4b Tension late wood, growing hyphae and enzymatically affected ladder-like perforation of the vessel, moderate degradation of the secondary wall warty layer. (Phanerochaete chrysosporium)
Figure 3c Normal late wood vessel, absent warty layer, dissolution of pits membranes in the vessel and ray parenchyma. (Ceriporiopsis subvermispora)
Figure 4c Vessel of tension late wood, partial degradation of pits membranes issuing into the ray parenchyma, moderate degradation of warty layer of the vessels secondary wall and partly delignified middle lamela. (Ceriporiopsis
SEM1 of sound normal and tension wood point out to untouched pits membranes of spring wood vessels (Figs. 1a and 2a ) and incomplete ladder like perforation in late wood vessels (3a and 4a). Figures 1b and 2b show the enzymatic dissolution of pits membranes in the spring wood vessels of normal and tension wood degraded by P. chrysosporium. In Figures 1c and 2c a less apparent decomposition of pits membranes in the both tension and normal beech wood can be seen. In Fig. 1c a resistant cell wall of the fiber with pattern of degradation in the middle lamella is aparent. SEM 3b (normal wood degraded by P. chrysosporium) shows the absence of warty layer in the secondary cell wall of the late wood vessel; Fig. 4b illustrates the ladder-like perforation of the late tension wood vessel with a moderate degradation of warty layer of the secondary cell wall in the vessel. 1
- for SEM and study of swelling kinetics of biodegraded wood (tensional and non-tensional) three specimens each with the weight loss approx. equal to mean of that for all biodegraded specimens were used, - the swelling kinetics has been examined following the method described in detail in (SOLÁR et al. 2001) - all experiments were carried out at the temperature of 20 oC,
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subvermispora) Figure 3c represents the vessel of the normal late wood degraded by C. subvermispora with the absence of warty layer, enzymatically damaged pits membranes of the vessel and adjacent ray parenchyma. In Figure 4c (degradation by C. subvermispora) only partial decomposition of pits membranes issuing into the ray parenchyma, and the released middle lamella of the vessel in tension late wood can be seen. Different permeability of normal and tension wood samples degraded by erosive and lignin selective fungus probably follows from different mode of action of each applied fungus represented by different pattern of the cell walls decomposition. Higher degree of dissolution of pits membranes and appearance of numerous erosions caused by erosive fungus P. chrysosporium increase porosity of wood, and explain the enhanced permeability of both samples of beech wood. On the other hand, reduced permeability of either beech wood samples after degradation by lignin selective strain of C. subvermispora might be due to enhanced swelling of their to high degree enzymatically delignified cell walls under conditions of determination (removal of lignin: approx. 10 % by P. chrysoWood structure and Properties ´02
sporium, and 20 % by C. subvermispora (SOLÁR et al. 2002)). A difference between the both sound and biodegraded beech wood samples dwells also in the intensity of wood/ distilled water interactions, expressed by the kinetics of their facial swelling (Figs. 5 and 6). Similar phenomenon was reported for interactions in the systems “sound (biodegraded) hornbeam wood/pulping liquors” (SOLÁR et al. 2001). 24
k=0,557 [s-1 ] k=0,207 [s-1 ]
Surface swelling [%]
22
1 1 - sound normal wood
20
3 - biodegraded byC. subvermispora
18
2 - biodegraded byP. chrysosporium
16 2
14
2
3
12
3
10 8 6
1
4
k=0,0073 [s-1 ]
2 0 0
60 120 180 240 300 360 420 480 540 600 660 720 780 84030000 900
Time [s]
Figure 5 Kinetics of surface swelling of sound and by P. chrysosporium and C. subvermispora degraded normal beech wood in the plain perpendicular to grain (medium: distilled water; t = 20 oC; initial moisture content of wood 2.5 %)
CONCLUSSIONS
24 k=0,1601 [s-1 ]
22
k=0,0866 [s-1 ]
2
20
2
Surface swelling [%]
18 1
16 14
3
3
12 10 1 - sound tensional wood
8
3 - biodegraded byC. subvermispora
6
2 - biodegraded byP. chrysosporium
4 2
1
k=0,0015 [s-1 ]
0 0
The final swelling of tension wood after degradation by P. chrysosporium exceeded apparently that of sound tension wood, and also that of biodegraded samples of normal wood. This phenomenon is in good agreement with extremely increased permeability of tension beech wood degraded by this fungus. The influence of medium-term degradation of normal and tension beech wood by lignin selective fungus C. subvermispora on the kinetics of surface swelling was less pronounced. This observation may result from rather lower degree of degradation of wood (expressed by the weight loss) and from its diminished axial permeability. Analyses of the swelling kinetics and permeability of sound and biodegraded samples of beech wood refer to positive effect of biodegradation on the acceleration of transport processes in wood, especially when erosive strain of white-rot fungus is used. Regarding the enhanced intensity of wood water interactions and the extent of final swelling especially in the case of fungaly degraded tension wood, we are of the opinion, that biodegradation contributed to equallisation of physical properties between normal and tension beech wood dependent on interactions with polar liquids.
60 120 180 240 300 360 420 480 540 600 660 720 780 84030000 900
Time [s]
Figure 6 Kinetics of surface swelling of sound and by P. chrysosporium a C. subvermispora biodegraded tension beech wood in the plain perpendicular to grain (medium: distilled water; t = 20 oC; initial moisture content of wood 2.8 %)
From the comparison of Figs. 5 and 6 a different rate of surface swelling of sound normal and tension beech wood is apparent. The intensity of swelling of normal wood was approx. 5 times higher as that of tension wood, and higher was also the final value of swelling after 8.5 h. of dipping in water. Biodegradation of both beech wood samples increased the kinetics of their initial pseudo-linear phase of swelling markedly (by almost two orders) and reduced the difference between the kinetics of normal and tension wood. In general, the extreme interactions of biodegraded beech wood samples with water resulted in nearly instant saturation of the cell walls in the specimens facial surface layers. Wood Structure and Properties ´02
Based on the achieved experimental data it may be stated, that a 30-day biodegradation of normal and tension beech wood by erosive fungus P. chrysosporium and lignin-selective fungus C. subvermispora caused the following structural alterations of examined wood samples accompanied by their modified physical properties : medium-term degradation of normal and tension beech wood by erosive strain of P. chrysosporium led to approximately doubled weight loss as compared to that caused by lignin-selective strain of fungus C. subvermispora, tension wood was more prone to biodegradation than normal wood, degradation of both normal and tension beech wood samples by the applied white-rot fungi modified their microstructure significantly and affected their axial permeability, degradation of tension wood by P. chrysosporium increased its permeability in dry and saturated state extremely, and the coefficient of permeability determined for the air dry material exceeded that determined for normal wood degraded by the same fungus, degradation of normal and tension beech wood samples by lignin selective fungus C. subvermispora reduced their axial permeability in dry and saturated states unexpectedly, biodegradation increased the swelling kinetics of normal and tension beech wood by almost two orders, especially when P. chrysosporium was used, 61
altered physical properties of fungally degraded normal and tension beech wood may positively influence its penetration with polar media and partly explain the increased kinetics of chemical delignification of fungally pretreated raw materials in the pulping processes.
REFERENCES SOLÁR, R. 2001: Vplyv parciálnej degradácie listnatých a ihličnatých drevín hubami bielej hniloby na prípravu vláknin a buničín. Vedecké štúdie 4/2001/A. Zvolen, TU vo Zvolene, 58 pp. SOLÁR R., KAČÍK F., REINPRECHT L. & MAMOŇOVÁ M. 2002: Zmeny základných zložiek bukového dreva v priebehu degradácie vybranými druhmi ligninovorných húb. In.: Chosen Processes at the Wood Processing. Zvolen, Technical University in Zvolen. MESSNER, K. & SREBOTNIK, E. 1994: Biopulping: An overview of developments in an environmentally safe papermaking technology. FEMS Microbiology Reviews 13:351364. SREBOTNIK,E. & MESSNER, K. 1994: Light microscopy of selective wood delignification by white-rot fungi using safranin and astra-blue differential staining. Appl. Environ. Microbiol. FEMS Microbiology Reviews 13:351364. SACHS, I. B. et al. 1989: Biomechanical pulping of aspen chips by Phanerochaete chrysosporium: Fungal growth pattern and effects on wood cell wals. Wood and Fiber Sci. 21(4):331342. SOLÁR, R. et al. 2001: Influence of hornbeam wood pretreratment by white-rot fungus Phanerochaete chrysosporium on the course of organosolv, kraft and neutral sulphite delignifications. Part. 2: Selected properties of the biodgraded material. Drevársky výskum 46(4): 922. LIESE, W. & SCHMID, R. 1966: Holz Roh Werkst. 24:454460. In: Fengel, D. Wegener, G.: Wood Chemistry,
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Ultrastructure and Reactions. 14. Microbial and Basidiomycetes Enzymatic Degradation. pp. 384. Walter de Gruyeter, Berlin-New York, 1984, 613 pp. BLANCHETTE, R. A. 1988: Resistance of hardwood vessels to degradation by white rot. Can. J. Bot. 66:18411847. NEČESANÝ, V. 1963: Holzforschung 17:5760. In: Fengel, D. Wegener, G.: Wood Chemistry, Ultrastructure and Reactions. Microbial and Enzymatic Dagradation pp. 385. Walter de Gruyeter, Berlin-New York, 1984, 613 pp. SCHMID, R. LIESE, W. 1964: Arch. Mikrobiol. 47:260276. In: Fengel, D. Wegener, G.: Wood Chemistry, Ultrastructure and Reactions. Microbial and Enzymatic Degradation pp. 385. Walter de Gruyeter, Berlin-New York, 1984, 613 pp. ERIKSSON, K. E. et al. 1980: Holzforschung 34:207213. In: Fengel, D.,Wegener, G.: Wood Chemistry, Ultrastructure and Reactions.14. Microbial and Enzymatic Degradation pp. 385. Walter de Gruyeter, Berlin-NewYork, 1984, 613 pp. SOLÁR, R. et al. 2000: Study of the overall alterations of hornbeam wood chips (Carpinus betulus L.) with emphasis on lignin in the pretreatment by white rot fungus Phanerochaete chrysosporium. Drevársky výskum 45(4):1932. REGINÁČ, L., ČOP, D. & ŠTEFKA, V. 1977: Zariadenie na skúšanie prieputnosti pórovitých materiálov najmä dreva pre kvapaliny a plyny. (Device for testing the permeability of porous materials with liquids and gases, especially of wood). Patent ČSFR, PV 5308-77 (11.8.1977) SOLÁR, R., KURJATKO, S. & REINPRECHT, L. 2001: Influence of hornbeam wood pretreatment by white-rot fungus Phanerochaete chrysosporium on the course of organosolv, sulphate and neutral sulphite delignifications. Part.1: Pulping and the selected pulps properties. Drevársky výskum 46(3):1120.
Acknowledgement This work was supported in part by Slovak Grant agency for science (grant No 1/7579/20).
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