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Effects of wood species and enzyme production on lignocellulose degradation during the biodegradation of three native woods by Trametes versicolor.

Abstract

This paper investigated effects of wood species and enzyme production on lignocellulose degradation during the biodegradation by white-rot fungus. Three native wood species, Weeping-willow (Salix babylonica), China-fir (Cunninghamia lanceolata) and Moso-bamboo (Phyllostachys pubescens), were degraded by the white-rot fungus Trametes versicolor B 1. China-fir woods exhibited lower weight, lignin, and cellulose losses than Weeping-willow or Moso-bamboo woods. Levels of the cellulase activity had no correlation with the degree of cellulose biodegradation. Enzymatic hydrolysis experiments of woods showed that the celluloses of undegraded woods have similarly high resistance to degradation and the resistance of cellulose to degradation decreased after the biodegradation by Trametes versicolor B 1. However, the resistance of China-fir cellulose decreased more slowly than the other woods. The result indicated the cellulose of China-fir wood was more resistant to white-rot fungal attack.

Biodegradation with white-rot fungi is a promising wood treatment process because of low energy requirement and mild environmental impact (Hakalaa et al. 2004). Since white-rot fungi exhibit different degradation rates for lignin and cellulose during biological treatment of different woods (Blanchette 1991), some wood species may not be good candidates for this process. Some studies attributed the differences to different ratios of syringyl (S) to guaiacyl (G) in wood lignin. Syringyl units are usually degraded preferentially than guaiacyl units by white-rot fungi (Faix et al. 1985, Martinez et al. 2001). However, there are no reported studies on the resistance of wood cellulose to biodegradation by white-rot fungi. Thus, it is necessary to study the relationship between cellulose degradation and the resistance of cellulose.

Enzyme expression in solid-substrate fermentation varies greatly with wood species (D'Souza et al. 1999). Several studies have reported enzyme production patterns of white-rot fungi incubated on the wood chip and malt-extract agar (Machuca and Ferraz 2001, Ferraz et al. 2003). However, few studies have evaluated enzyme production during solid fermentation using wood as the sole nutrient source. Therefore, measuring enzymes production on wood alone will aid in understanding the enzymatic mechanisms of wood biodegradation.

The native wood species, China-fir (Cunninghamia lanceolata), Moso-bamboo (Phyllostachys pubescens), and Weeping-willow (Salix babylonica) are widely used in the Chinese wood industry. Taxonomically, the three wood species are respectively gymnosperm, monocotyledon and dicotyledon. This study investigated the effects of wood species and enzyme production on lignocellulose degradation by white-rot fungus, Trametes vesicolor. Cellulases and ligninolytic enzymes in extracts from degraded woods were compared during the biodegradation. Lignin and cellulose contents in the degraded woods were also measured.

Methods and materials Wood preparation and biodegradation experiments

Three wood species, China-fir (Cunninghamia lanceolata), Weeping-willow (Salix babylonica) and Moso-bamboo (Phyllostachys pubescens), were obtained from Wuhan, China. All woods, free of bark, were air-dried and ground to pass through a 0.9 mm screen and then ovendried at 60 [degrees]C for 3 days.

Trametes versicolor B 1 was isolated from a hardwood in Shenlongjia nature reserve (Hubei, China). The isolate was grown on potato-dextrose agar (PDA) at 25 [degrees]C for 10 days before cutting inoculating plugs. Biodegradation experiments were carried out in 125-ml Erlenmeyer flasks with 5 g ground wood and 10ml water. Flasks were sterilized in an autoclave for 20 minutes at 121 [degrees]C and aseptically inoculated with a plug cut from the margins of the PDA fungal culture. Noninoculated wood was used as control. Cultures were incubated statically at 25 [degrees]C for periods varying from 10 to 120 days.

Chemical analyses of wood

At the end of each incubation period, the wood culture was extracted with 45ml of 50 mM sodium acetate buffer (pH 5.0) supplemented with 0.01 percent (weight/volume, w/v) Tween 80 on a rotary shaker (150 rpm) at 25 [degrees]C for 6 hours. Extracts were filtered through filter paper and assayed to determine the enzyme activities. Extracted wood was dried at 60 [degrees]C for 3 days and then weighed to determine weight loss. Lignin and cellulose were determined according to procedures of AOAC (official methods of analysis of the Association of Official Analytical Chemists) (Horwitz 1980).

Enzyme assays

Laccase activity was determined with 2,2'-azino-bis(3-ethylbenzthiazoline-6-Sulfonate) (ABTS) (Steffen et al. 2000). Activity of manganese peroxidase (MnP) was measured at 270 nm by following the formation of [Mn.sup.3+]-malonate complexes at pH 4.5 in 50 mM sodium malonate buffer (Hakalaa et al. 2005). Lignin-peroxidase activity was not assayed because of interference by extracts of the solid-substrate fermentation (Machuca and Ferraz 2001).

Trametes versicolor produces a multicomponent cellulase system, consisting of endoglucanase (EC 3.2.1.4), cellobiohydrolase (EC 3.2.1.91), and [beta]-glucosidase (EC 3.2.1.21) (Cai et al. 1999). The activities of all three cellulases and total cellulase were determined in this study. Endoglucanase, cellobiohydrolase and total cellulase were assayed in 50 mM sodium acetate buffer (pH 4.8) at 50 [degrees]C by release of reducing sugars measured with 3,5-dinitrosalicylic acid (DNS) reagent, using the appropriate substrates respectively: carboxymethycellulose-Na (CMC-Na), microcrystalline cellulose and filter paper strip (Whatman No. 1) (Mata and Savoie 1998, Cai et al. 1999, Wang et al. 2004). P-Nitrophenyl-[beta]-D- glucopyranoside (p-NPG) was used in assays for [beta]-glucosidase activity (Ferraz et al. 2003).

Enzymatic hydrolysis experiments

In order to investigate the resistance of cellulose to fungal biodegradation, cellulolytic hydrolysis of degraded and undegraded woods were performed. The hydrolysis rates of wood cellulose were used as indicators of the resistance because the resistance cannot be measured directly. The hydrolysis assay consisted of incubating 2.5 percent (w/v) wood substrate with cellulase (10 FPU/g wood substrate) in 50 mM sodium acetate buffer (pH 4.8) at 50 [degrees]C for 24 hours. Cellulase was obtained from Sigma. After 24 hours, the release of reducing sugars was determined with the DNS reagent (Cai et al. 1999). Hydrolysis rate of cellulose in woods was calculated as the percent of reducing sugars produced per gram of wood cellulose.

[FIGURE 1 OMITTED]

Results and discussion

Chemistry analysis of degraded woods

Weeping-willow and Moso-bamboo wood exhibited higher weight, lignin and cellulose losses than China-fir wood (Fig. 1). On the 120th day of biodegradation, the highest weight (45.13%), lignin (58.37%) and cellulose (47.52%) losses were obtained in the Weeping-willow wood. The rate of cellulose degradation in China-fir woods was very low at early stages (3.25 %, the 30th day), and increased only after 30-day degradation. Although celluloses of Moso-bamboo and Weeping-willow woods were also barely degraded during the first 10 days, the rates of cellulose degradation increased rapidly after the 10th day. Angiosperm wood lignin was more easily degradable than gymnosperm wood lignin by white-rot fungi because of a very high concentration of G units in the gymnosperm woods (Faix et al. 1985). Thus, Trametes versicolor B 1 caused lower lignin loss of China-fir wood, a gymnosperm stem, than Weeping-willow and Moso-bamboo woods, which were all angiosperm stems.

Enzymes production during the biodegradation

Cellulases and ligninolytic enzymes were extracted and measured to study the effect of enzyme production on lignocellulose degradation (Table 1). Although cellulose loss on China-fir wood was significantly lower than other woods, there were no significant differences in total cellulase activities on China-fir, Weeping-willow, and Moso-bamboo woods by the paired t-test on the data from Table 1 at 99 percent confidence level. The levels of endoglucanases activity were similar on all woods and reached its peaks at 30 to 60 days, while the level of [beta]-glucosidase activity was higher on the Moso-bamboo woods than Weeping-willow and China-fir woods. The levels of [beta]-glucosidase activity produced by Trametes versicolor B 1 were similar over the entire decay period for each kind of wood. Cellobiohydrolase activity varied greatly during the whole period of decay and disappeared at the advanced stages of decay. There was no correlation between the cellulose degradation and cellulases activity during the biodegradation of China-fir, Weeping-willow, and Mosobamboo woods.

Trametes versicolor B1 produced a high level of manganese peroxidase (MnP) on Moso-bamboo woods. MaP reached its highest level of activity at the early decay stages (before 30 days) and disappeared on the 90th or 120th day. Laccase activity reached its highest level on the 10th day and then decreased. The levels of laccase activity were usually lower in the China-fir wood than in the Weeping-willow and Moso-bamboo wood. Interestingly, the two ligninolytic enzymes showed similar profiles with low activity at advanced stages of decay, when the low rates oflignin degradation were obtained (Fig. 1, Table 1).

Effect of wood species on cellulose degradation

With the same substrate concentration, cellulase load and reaction condition, enzymatic hydrolysis rate of wood celluloses should be inversely correlated to the resistance of wood cellulose to the degradation. Thus, enzymatic hydrolysis rates of degraded and undegraded woods were measured invitro (Fig. 2) and used as an indicator of the resistance of wood cellulose to biodegradation by white-rot fungi. Undegraded wood showed very low hydrolysis rate, which indicated natural wood cellulose was highly resistant to the biodegradation. In all undegraded woods, the Weeping-willow wood showed a little higher cellulose hydrolysis rate (5.30%) than Mosobamboo (1.97%) and China-fir woods (1.46%). Degraded wood showed increase of cellulose hydrolysis rates, which indicated the resistance of cellulose to degradation decreased after the biodegradation. However, the resistance of China-fir cellulose decreased more slowly. The degraded China-fir wood showed only 7.5 percent hydrolysis rate at 120 days, while Moso-bamboo and Weeping-willow woods showed 34.5 percent and 34.8 percent hydrolysis rates.

[FIGURE 2 OMITTED]

It is well known that the cellulases during wood biodegradation are too large to penetrate the cell walls and lignin serves as a barrier to cellulose. Thus, celluloses of all undegraded woods showed high resistance to degradation. Weeping-willow and Moso-bamboo woods underwent higher lignin losses and became more porous during the decay by white-rot fungi, and then cellulases can penetrate further into cell walls. China-fir underwent lower lignin losses and left the most lignin, which could negatively impact the penetration of cellulases. So the resistance of China-fir cellulose to the fungal degradation was significantly lower than the other wood species and thus impacts the rates of cellulose degradation by white-rot fungi.

Conclusions

Trametes versicolor B1 exhibited different degradation abilities of lignin and cellulose in China-fir, Weeping-willow, and Moso-bamboo woods. China-fir cellulose was more resistant to white-rot fungal degradation than Weeping-willow and Moso-bamboo cellulose. The level of the cellulase activity had no correlation with the extent of cellulose removal.

Literature cited

Blanchette, R.A. 1991. Delignification by wood-decay fungi. Annu. Rev. Phytopathol. 29:381-403.

Cai, Y.J., S.J. Chapman, J.A. Buswell, and S.T. Chan. 1999. Production and distribution of endoglucanase, cellobiohydrolase, and betaglucosidase of the cellulolytic system of Volvariella volvacea, the edible straw mushroom. Appl. Environ. Microbiol. 65(2): 553-559.

D'Souza, T.M., C.S. Merritt, and C.A. Reddy. 1999. Lignin-modifying enzymes of the white rot basidiomycete Ganoderma lucidum. Appl. Environ. Microbiol. 65(12):5307-5313.

Faix, O., M.D. Mozuch, and T.K. Kirk. 1985. Degradation of gymnosperm (Guaiacyl) vs. angiosperm (syringyl/guaiacyl) lignins by Phanerochaete chrysosporium. Holzforschung 39:203-208.

Ferraz, A., A.M. Cordova, and A. Machuca. 2003. Wood biodegradation and enzyme production by Ceriporiopsis subvermispora during solidstate fermentation of Eucalyptus grandis. Enzyme Microb. Tech. 32(1):59-65.

Hakalaa, T.K., T. Lundella, S. Galkina, P. Maijalaa, N. Kalkkinenb, and A. Hatakkaa. 2005. Manganese peroxidases, laccases and oxalic acid from the selective white-rot fungus Physisporinus rivulosus grown on spruce wood chips. Enzyme Microb. Tech. 36(4):461-468.

--, P. Maijala, J. Konnb, and A. Hatakkaa. 2004. Evaluation of novel wood-rotting polypores and corticioid fungi for the decay and biopulping of Norway spruce (Picea abies) wood. Enzyme Microb. Tech. 34(3-4):255-263.

Horwitz, W. (Ed). 1980. Official methods of analysis of the Assoc. of Official Analytical Chemists. Assoc. of Official Analytical Chemists, Washington.

Machuca, A. and A. Ferraz. 2001. Hydrolytic and oxidative enzymes produced by white- and brown-rot fungi during Eucalyptus grandis decay in solid medium. Enzyme Microb. Tech. 29:386-391.

Martinez, A.T., S. Camarero, A. Gutierrez, P. Bocchini, and G.C. Galletti. 2001. Studies on wheat lignin degradation by Pleurotus species using analytical pyrolysis. J. of Analytical and Applied Pyrolysis 5859:401-411.

Mata, G. and J.M. Savoie. 1998. Extracellular enzyme activities in six Lentinula edodes strains during cultivation in wheat straw. World J. Microbiol. Biotechnol. 14(4):513-519.

Steffen, K.T., M. Hofrichter, and A. Hatakka. 2000. Mineralisation of 14C-labelled synthetic lignin and ligninolytic enzyme activities of litter-decomposing basidiomycetous fungi. Appl. Microbiol. and Biotech. 54(6):819-825.

Wang, L.S., Y.Z. Zhang, H. Yang, and P.J. Gao. 2004. Quantitative estimate of the effect of cellulase components during degradation of cotton fibers. Carbohydr. Res. 339(4):819-824.

The authors are, respectively, Postdoctoral Researcher, Student, Professor, and Postdoctoral Researcher, College of Life Sci. and Technology, Huazhong Univ. of Sci. and Technology, Wuhan, P.R. China (imerhust@mail.hust.edu.cn, china_lovelylily@hotmail.com, hongbo.fish@gmail.com, h_y_huang0879@sina.com). This paper was received for publication in January 2007. Article No. 10299.
Table 1.--Production of cellulases and ligninolytic enzymes by Trametes
versicolor B1 grown on China-fir (Cunninghamia lanceolata),
Weeping-willow (Salix babylonica) and Moso-bamboo (Phyllostachys
pubescens) woods.

Enzymatic activity Decay time (days)
(IU/g) 10 20

 Weeping-willow (Salix babylonica)

Total cellulase 0.51 [+ or -] 0.02 0.15 [+ or -] 0.02
Endoglucanase 0.40 [+ or -] 0.01 0.77 [+ or -] 0.08
Cellobiohydrolase 0.60 [+ or -] 0.05 0
[beta]-glucosidase 0.22 [+ or -] 0.01 0.53 [+ or -] 0.05
Laccase 546 [+ or -] 18 76 [+ or -] 8
MnP 22 [+ or -] 1 33 [+ or -] 2

 China-fir (Cunninghamia lanceolata)

Total cellulase 0.24 [+ or -] 0.01 0.36 [+ or -] 0.04
Endoglucanase 0.06 [+ or -] 0.00 0.31 [+ or -] 0.03
Cellobiohydrolase 0.0 0.34 [+ or -] 0.06
[beta]-glucosidase 0.21 [+ or -] 0.01 0.36 [+ or -] 0.01

Laccase 117.4 [+ or -] 0.4 0.50 [+ or -] 0.05
MnP 27 [+ or -] 1 24 [+ or -] 5

 Moso-bamboo (Phyllostachys pubescens)

Total cellulase 0.18 [+ or -] 0.02 0.27 [+ or -] 0.02
Endoglucanase 0.30 [+ or -] 0.03 0.60 [+ or -] 0.06
Cellobiohydrolase 0.06 [+ or -] 0.00 0

[beta]-glucosidase 1.83 [+ or -] 0.15 0.65 [+ or -] 0.04
Laccase 231 [+ or -] 20 94 [+ or -] 11
MnP 148 [+ or -] 2 786 [+ or -] 13

Enzymatic activity Decay time (days)
(IU/g) 30 40

 Weeping-willow (Salix babylonica)

Total cellulase 0.21 [+ or -] 0.01 0.10 [+ or -] 0.01
Endoglucanase 0.83 [+ or -] 0.01 0.77 [+ or -] 0.07
Cellobiohydrolase 0.55 [+ or -] 0.01 0.06 [+ or -] 0.03
[beta]-glucosidase 0.46 [+ or -] 0.01 0.47 [+ or -] 0.05
Laccase 152 [+ or -] 16 76 [+ or -] 10
MnP 52 [+ or -] 2 31 [+ or -] 2

 China-fir (Cunninghamia lanceolata)

Total cellulase 0.23 [+ or -] 0.02 0.13 [+ or -] 0.01
Endoglucanase 0.93 [+ or -] 0.07 1.05 [+ or -] 0.09
Cellobiohydrolase 0.21 [+ or -] 0.01 0
[beta]-glucosidase 0.57 [+ or -] 0.06 0.52 [+ or -] 0.02

Laccase 35 [+ or -] 3 27 [+ or -] 1
MnP 11.2 [+ or -] 0.1 10 [+ or -] 1

 Moso-bamboo (Phyllostachys pubescens)

Total cellulase 0.09 [+ or -] 0.01 0.17 [+ or -] 0.03
Endoglucanase 0.46 [+ or -] 0.04 0.22 [+ or -] 0.02
Cellobiohydrolase 0.14 [+ or -] 0.08 0.20 [+ or -] 0.01

[beta]-glucosidase 1.35 [+ or -] 0.04 1.36 [+ or -] 0.07
Laccase 78 [+ or -] 3 104 [+ or -] 10
MnP 569 [+ or -] 30 459 [+ or -] 40

Enzymatic activity Decay time (days)
(IU/g) 50 60

 Weeping-willow (Salix babylonica)

Total cellulase 0.21 [+ or -] 0.01 0.04 [+ or -] 0.01
Endoglucanase 0.75 [+ or -] 0.05 0.61 [+ or -] 0.06
Cellobiohydrolase 0.26 [+ or -] 0.02 0.04 [+ or -] 0.02
[beta]-glucosidase 0.37 [+ or -] 0.03 0.58 [+ or -] 0.03
Laccase 91 [+ or -] 3 77 [+ or -] 7
MnP 24 [+ or -] 2 16 [+ or -] 3

 China-fir (Cunninghamia lanceolata)

Total cellulase 0.25 [+ or -] 0.01 0.08 [+ or -] 0.01
Endoglucanase 0.83 [+ or -] 0.02 0.48 [+ or -] 0.05
Cellobiohydrolase 0.18 [+ or -] 0.01 0.03 [+ or -] 0.00
[beta]-glucosidase 0.30 [+ or -] 0.01 0.26 [+ or -] 0.02

Laccase 23 [+ or -] 2 45 [+ or -] 3
MnP 11 [+ or -] 1 6.7 [+ or -] 0.4

 Moso-bamboo (Phyllostachys pubescens)

Total cellulase 0.09 [+ or -] 0.05 0.24 [+ or -] 0.02
Endoglucanase 0.70 [+ or -] 0.07 0.37 [+ or -] 0.02
Cellobiohydrolase 0.37 [+ or -] 0.01 0.14 [+ or -] 0.01

[beta]-glucosidase 1.10 [+ or -] 0.08 1.53 [+ or -] 0.15
Laccase 35 [+ or -] 3 38 [+ or -] 4
MnP 301 [+ or -] 16 223 [+ or -] 18

Enzymatic activity Decay time (days)
(IU/g) 90 120

 Weeping-willow (Salix babylonica)

Total cellulase 0.12 [+ or -] 0.04 0.08 [+ or -] 0.01
Endoglucanase 0.55 [+ or -] 0.02 0.16 [+ or -] 0.02
Cellobiohydrolase 0 0.15 [+ or -] 0.04
[beta]-glucosidase 0.76 [+ or -] 0.04 0.79 [+ or -] 0.03
Laccase 26 [+ or -] 2 78 [+ or -] 3
MnP 0 0

 China-fir (Cunninghamia lanceolata)

Total cellulase 0.10 [+ or -] 0.01 0.07 [+ or -] 0.01
Endoglucanase 0.68 [+ or -] 0.11 0.21 [+ or -] 0.02
Cellobiohydrolase 0 0
[beta]-glucosidase 0.44 [+ or -] 0.01 0.47 [+ or -] 0.03

Laccase 38 [+ or -] 4 48 [+ or -] 6
MnP 2.6 [+ or -] 1.4 0

 Moso-bamboo (Phyllostachys pubescens)

Total cellulase 0.07 [+ or -] 0.01 0.18 [+ or -] 0.02
Endoglucanase 0.60 [+ or -] 0.02 0.42 [+ or -] 0.01
Cellobiohydrolase 0 0

[beta]-glucosidase 2.04 [+ or -] 0.15 1.87 [+ or -] 0.14
Laccase 38.8 [+ or -] 0.1 52 [+ or -] 3
MnP 97 [+ or -] 9 0
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Author:Yu, Hong-Bo; Li, Li; Zhang, Xiao-Yu; Huang, Hui-Yan
Publication:Forest Products Journal
Geographic Code:1USA
Date:Apr 1, 2008
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