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Evaluation of biological pretreatment with white rot fungi for the enzymatic hydrolysis of bamboo culms
ARTICLE IN PRESS
International Biodeterioration & Biodegradation 60 (2007) 159–164 www.elsevier.com/locate/ibiod
Evaluation of biological pretreatment with white rot fungi for the enzymatic hydrolysis of bamboo culms Xiaoyu Zhang, Hongbo Yu, Huiyan Huang, Youxun Liu College of Life Science and Technology, Huazhong University of Science and Technology, 430074 Wuhan, P.R. China Received 27 November 2006; received in revised form 9 February 2007; accepted 9 February 2007 Available online 25 April 2007
Abstract Biological pretreatment with white rot fungi has shown potential for improving enzymatic hydrolysis of wood and grass. In this study, 34 isolates of white rot fungi were screened for the biological pretreatment of bamboo culms (Phyllostachys pubescence). Echinodontium taxodii 2538 and Trametes versicolor G20 were selected for further evaluation of pretreatment because they caused high lignin loss ð420%Þ and high selectivity value of lignin degradation ð42Þ after the 4-week biodegradation. Fermentable sugar yield of bamboo culms pretreated with these two fungi through enzymatic hydrolysis increased with increasing pretreatment time. Sugar yield of bamboo culms pretreated with T. versicolor G20 and E. taxodii 2538 increased 5.15-fold and 8.76-fold, respectively, after 120-day pretreatment. FTIR analysis showed that E. taxodii 2538 preferentially degraded the lignin of bamboo culms. The pretreated bamboo culms showed significant increase of initial adsorption capacity to cellulase (4.20-fold and 6.66-fold for T. versicolor G20 and E. taxodii 2538, respectively, after 120 days) and decrease of lignin content (12.00% and 29.14% for T. versicolor G20 and E. taxodii 2538, respectively, after 120 days) with increasing pretreatment time. Initial adsorption capacity and lignin content of bamboo culms were correlated to fermentable sugar yield. Scientific relevance This paper focused on the biodegradation and pretreatment of bamboo culms with white rot fungi. Bamboo culms pretreatment with white rot fungi was evaluated firstly for energy convention of lignocellulose. This paper studied effects of lignin content and initial adsorption capacity on enzymatic hydrolysis also. r 2007 Elsevier Ltd. All rights reserved. Keywords: Trametes versicolor; Echinodontium taxodii; Bamboo culms; Biological pretreatment; Enzymatic hydrolysis; Lignin; Adsorption
1. Introduction Bamboos are perennial woody grasses belonging to the Gramineae family and Bambuseae subfamily and distribute widely in Asia (Lee et al., 2001; Scurlock et al., 2000; Vu et al., 2004). China is a major bamboo-producing country and has about 300 species in 44 genera, occupying 3% of total forest area (Scurlock et al., 2000). Bamboo has been widely used as the feedstock of paper, textile, food, construction and reinforcing fibers. Recently, some studies indicated bamboo culms could be exploited as the feedstock of biomass energy, such as ethanol and methanol (Tsuda et al., 1998; Kobayashi et al., 2004; Scurlock et al., 2000). Corresponding author. Tel.: +86 27 8779 2128; fax: +86 27 8779 2108.
E-mail address: [email protected] (X. Zhang). 0964-8305/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2007.02.003
It is necessary to degrade the lignin by the pretreatment for energy production from bamboo culms since the lignin network prevents enzymatic treatment of bamboo culms and release of fermentable sugars. White rot fungi are the only known organisms efficiently removing lignin from the plant cell walls in the world (Hakalaa et al., 2005). Biological pretreatment of lignocellulose with white rot fungi is a promising technology for energy production from lignocellulose because of the advantages of low energy requirement and mild environmental conditions (Hakalaa et al., 2004; Sun and Cheng, 2002). Some studies indicated the pretreatment with white rot fungi could reduce lignocellulose recalcitrance to enzymatic hydrolysis and improve the technological process of energy production from wood and grass (Itoh et al., 2003; Taniguchi et al., 2005). Currently, some physical and chemical pretreatment
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have been used for the enzymatic hydrolysis of bamboo culms in the energy production (Tsuda et al., 1998; Kobayashi et al., 2004). However, biological pretreatment of bamboo culms with white rot fungi had been neglected. Bamboo culms have differences from wood and grass in chemical constituents and fiber characteristic (Scurlock et al., 2000). Accordingly, it is necessary to evaluate the pretreatment with white rot fungi for enzymatic hydrolysis of bamboo culms. In this study, 34 isolates of white rot fungi were screened for the biological pretreatment of Moso bamboo culms (Phyllostachys pubescence) by the ability to degrade bamboo lignin and selectivity value of lignin degradation. Pretreatments of bamboo culms with Echinodontium taxodii 2538 and Trametes versicolor G20 which were selected according to the results of the first screening step were evaluated in more details including saccharification, lignin content and initial adsorption capacity. Biodegradation patterns of the pretreated bamboo culms were also characterized by FTIR analysis.
2. Materials and methods 2.1. Microorganisms and isolation Most of the fungi used in the evaluation were isolated from basidiocarps of white rot fungi which were collected in Shenlongjia nature reserve (Hubei, China) and caused great decay of host plant. Other fungi were obtained from our laboratory. Isolates were recovered by placing small pieces of fruit body plectenchyma aseptically or decayed wood under the fruit body on potato dextrose agar (PDA) containing streptomycin ð50 mg l1 Þ to prevent growth of bacteria. Stock cultures of fungi were maintained on PDA slants at 4 1C. The species were listed in Table 1. Fungi from Shenlongjia nature reserve were identified based on their macro and microscopic morphological characteristics by Dr. Yucheng Dai and Dr. Kari T. Steffen. E. taxodii 2538 were further identified by comparing the nucleotide sequences of 5.8S rRNA gene with those of related strains in GenBank. The GenBank accession number of E. taxodii 2538 was EF422215.
Table 1 Percent losses of different components of Moso bamboo (Phyllostachys pubescence) culms after 4-week pretreatment by 34 white rot fungi Species and strainsa
Antrodiella zonata 5893 Boletus sp. P44b Collybia sp. 2813 Daedaleopsis confrgosa 246 Echinodontium taxodii 2538d Flammulina velutipes WFHb Fomes fomentarius 248 Ganoderma australe P17c
Selectivity Component loss (%) value Weight Lignin Cellulose Hemicellulose
Table 1 (continued ) Species and strainsa
Selectivity Component loss (%) value Weight Lignin Cellulose Hemicellulose
Ganoderma lipsisense 245 Ganoderma lucidum En4 Ganoderma sp. En1b Ganoderma sp. En3 Ganoderma sp. En5 Grifola umbellata P64b Hygrocybe punicea 258 Pachykytospora papyracea 5865 Perenniporia sp. 5814 Pleurotus ostreatus Bp2b Polyporus mongolius 2410 Rigidoporus crocatus 5899 Russula sp. 2416 Schizopora flavipora 5951 Trametes gibbosa 5955 Trametes ochracea 243 Trametes ochracea 5930 Trametes ochracea 5897 Trametes ochracea P58 Trametes versicolor CD1 Trametes versicolor G20 Trametes sp. Yhb-5 Trichaptum abietinum 247 Trichaptum biforme 2526 Trichaptum sp. P39 Tyromyces chioneus 5877
2.74
10.78
16.25 5.92
19.31
0.82
12.10
10.56 12.83
15.16
1.15 2.30 0.58 1.11
6.76 8.36 2.59 8.26
5.10 15.72 1.72 10.01
4.45 6.84 2.99 9.00
17.30 9.95 0.05 13.85
1.68
3.13
6.59
3.93
0.60
None
None
None None
None
6.61 0.61
2.51 5.17
8.66 2.67
1.31 4.38
3.36 12.59
3.88
9.01
13.07 3.37
16.13
None
None
None None
None
None None
None None
None None None None
None None
1.45
11.72
13.40 9.22
24.77
0.86
15.49
13.26 15.49
27.24
2.90
11.05
18.20 6.27
21.88
0.54
10.23
5.50
10.23
25.06
1.73
15.21
18.63 10.79
29.22
4.25
6.62
9.14
1.75
2.61
13.63
24.42 9.35
18.89
2.12 1.25
7.37 10.14
13.16 6.22 13.56 10.86
9.78 21.08
1.48
11.04
12.54 8.48
32.70
0.86 1.85
9.19 3.18
7.60 1.44
25.78 13.29
2.15
8.81 0.78
a
Fungi from shenlongjia nature reserve were identified by Dr. Yucheng Dai and Dr. Kari T. Steffen. b Obtained from our laboratory. c Obtained from YiChang, Hubei province, and identified by Dr. Yucheng Dai. d The GenBank accession number: EF422215.
1.52
6.34
5.50
3.63
17.74
6.00 0.64 1.65
2.85 11.28 8.74
0.24 8.12 8.74
0.04 12.75 5.30
12.14 20.54 28.58
14.8
10.58
24.28 1.64
28.46
2.2. The first screening step
0.81
2.27
3.14
3.88
4.82
6.24
0.50
3.12
0.50
0.50
1.23
9.11
7.49
6.11
18.60
Moso bamboo (Phyllostachys pubescence) culms from Wuhan were ground to pass through a 0.9 mm screen and then were kiln-dried at 60 1C for three days. In order to select the best strains for the subsequent pretreatment evaluation, 34 fungi were screened by lignin-degrading ability and selectivity value of lignin degradation. The experiments were carried out in Petri dishes with 5 g bamboo culms and 10 ml of synthetic
ARTICLE IN PRESS X. Zhang et al. / International Biodeterioration & Biodegradation 60 (2007) 159–164 medium with the following composition per liter: MgCl2 0.01 g, KH2PO4 0.002 g, CaCl2 0.0005 g and yeastrel 0.05 g. Petri dishes were used as the decay chambers. Film was wrapped around Petri dishes to act as a barrier against moisture loss and contamination. Small perforations were made to the film to avoid moisture condensation and allow ventilation of chambers. All isolates of white rot fungi were grown on PDA plate at 25 1C for 10 days before cutting inoculating plugs. Petri dishes were sterilized in the autoclave for 20 min at 121 1C and aseptically inoculated with a plug cut from the margins of the PDA culture. After Petri dishes were incubated at 25 1C for four weeks, the mycelium covering the bamboo in Petri dishes was gently removed. The decayed bamboo culms were dried at 60 1C for three days and then weighted to calculate weight losses based on the initial and final dry weights. Lignin and cellulose were determined according to procedures of AOAC (1980) (Horwirtz, 1980). Hemicellulose was determined by the difference between neutral detergent fiber (NDF) and acid detergent fiber (ADF) (Rezaeian et al., 2005). The selectivity value of lignin degradation was calculated as lignin loss/ cellulose loss ratio.
2.3. Bamboo culms pretreatment with white rot fungi The biological pretreatments with E. taxodii 2538 and T. versicolor G20 were carried out in 250-ml Erlenmeyer flasks with 10 g ground bamboo culms and 20 ml distilled water. Flasks were sterilized in the autoclave for 20 min at 121 1C and aseptically inoculated with a plug cut from the margins of the PDA culture. Cultures were maintained statically at 25 1C for 30, 60, 90 and 120 days and then dried at 60 1C for three days removing mycelium covering the bamboo. All the cultures were grown in triplicate. Non-inoculated bamboo was used as control.
2.4. FTIR analysis of biodegradation patterns Bruker Vertex 70 infrared spectrophotometer was used to investigate biodegradation patterns of bamboo culms pretreated with E. taxodii 2538 and T. versicolor G20. The dried bamboo samples were mixed with KBr of spectroscopic grade and made in the form of pellets at pressure of about 1 MPa. The pellets were about 10 mm in diameter and 1 mm thickness. Peak height and area were determined by Omnic software and relative changes in the intensity of lignin/carbohydrate characteristic bands were analyzed by Pandeya’s method (Pandeya and Pitmanb, 2003).
2.5. Enzymatic hydrolysis Enzymatic hydrolysis experiments of bamboo culms pretreated with E. taxodii 2538 and T. versicolor G20 were performed to investigate effect of biological pretreatment. Hydrolysis was carried out at 2.5% bamboo substrate concentration in 50 mM sodium acetate buffer (pH 4.8) with cellulase (20 FPU g1 substrate) at 50 1C. Cellulase was obtained from Sigma. After 6, 12, 24, 48, 72, 96, 120 h, the release of fermentable sugars was determined with the DNS (3,5-dinitrosalicylic acid) reagent (Behera et al., 1996; Mata and Savoie, 1998).
2.6. Adsorption measurements An enzyme loading of 20 FPU g1 bamboo substrate was used to investigate initial adsorption capacity to cellulase. Adsorption reaction was performed as described by Mooney et al. (1998) in 50 mM sodium acetate buffer (pH 4.8) for 90 min at 4 1C and then for 10 min at 50 1C. Protein in the centrifugate was measured using the Bradford protein assay after centrifugation at 10 000 rpm for 5 min (Mooney et al., 1999). Adsorbed cellulase protein was calculated based on the difference between the amount of initial protein and unadsorbed protein in the centrifugate. Initial adsorption capacity of bamboo substrate was calculated as percent ratio of adsorbed protein/initial protein.
161
3. Results and discussion 3.1. The first screening step The weight and component losses of bamboo culms pretreated by 34 fungi were listed in Table 1. All fungi caused the delignification of bamboo culms except for four fungi which were not able to grow in the bamboo culms. In most cases, white rot fungi caused higher hemicellulose loss than other component losses. Although Trametes ochracea 243 had the greatest degrading ability in all tested fungi after the 4-week of cultivation, it was not suitable for bamboo pretreatment because it simultaneously degraded cellulose and lignin (selectivity valueo1). Low selectivity value meant relatively high cellulose loss during the biological pretreatment. Thus, the selectivity value of lignin degradation and lignin loss were used to screen white rot fungi for biological pretreatment. Eleven strains had great selective lignin-degrading ability (selectivity value42). Among these fungi, E. taxodii 2538 and T. versicolor G20 caused the greatest lignin loss. Therefore, E. taxodii 2538 and T. versicolor G20 were selected to further study for evaluation of bamboo culms pretreatment. 3.2. Evaluation of biodegradation patterns Biodegradation patterns of bamboo culms during the biological pretreatment with E. taxodii 2538 and T. versicolor G20 were evaluated by FTIR analysis. Fig. 1A and B showed the FTIR spectra of bamboo culms pretreated with two fungi for different days. Spectrum of bamboo culms pretreated with E. taxodii 2538 revealed a proportional decrease in intensity of lignin peak at 1510 cm1 which was assigned for aromatic skeletal in lignin with prolonged pretreatment (Pandeya and Pitmanb, 2003). There were little changes at 1510 cm1 peak of bamboo culms pretreated with T. versicolor G20 although these two fungi had similar lignin-degrading ability. In order to fully understand biodegradation patterns of bamboo culms, we made a detailed FTIR spectroscopic analysis based on Pandeya’s analysis method (Pandeya and Pitmanb, 2003). Table 2 showed relative changes in the intensities of lignin peaks at 1510 cm1 against carbohydrate peaks at 1734, 1378, 1163 and 898 cm1 which were calculated by peak heights and areas. These four carbohydrate peaks were assigned, respectively, for unconjugated CQO in xylans, C–H deformation in cellulose and hemicellulose, C–O–C vibration in cellulose and hemicellulose and C–H deformation in cellulose (Pandeya and Pitmanb, 2003). There was significant decrease in the lignin/carbohydrate ratio of bamboo pretreated by E. taxodii 2538 with increasing pretreatment time, which indicated the fungus had great selective lignin-degrading ability. The lignin/carbohydrate ratio of bamboo pretreated with T. versicolor G20 increased at advanced stages of pretreatment although it decreased at the early stage (before 60 days). These results indicated that T. versicolor G20 preferentially degrade lignin only at the
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(4) (2)
(1)
(1)
(3)
(4) (5)
e Absorbance
Absorbance
(3)
(5)
e d
d c
c
b
b
a 1800
(2)
a 1600
1400
1200
Wavenumbers
1000
800
1800
1600
1400
1200
1000
800
Wavenumbers (cm-1)
(cm-1)
Fig. 1. FTIR spectra of bamboo culms pretreated with Trametes versicolor G20 (A) and Echinodontium taxodii 2538 (B) for different days: (a) control; (b) pretreated for 30 days; (c) pretreated for 60 days; (d) pretreated for 90 days; (e) pretreated for 120 days. (1) 1734 cm1 , (2) 1510 cm1 , (3) 1378 cm1 , (4) 1163 cm1 , (5) 898 cm1 .
Table 2 Ratios of the intensity of the lignin associated band with carbohydrate bands for pretreated samples Pretreatment Relative intensities of aromatic skeletal vibration (I 1510 ) against typical bands for carbohydrates time (days) I 1510 =I 1734 Control 0 0.80 (0.57) Trametes versicolor G20 30 0.64 (0.44) 60 0.33 (0.20) 90 0.45 (0.27) 120 0.70 (0.52) Echinodontium taxodii 2538 30 0.53 (0.37) 60 0.48 (0.31) 90 0.38 (0.25) 120 0.26 (0.17)
I 1510 =I 1378
I 1510 =I 1163
I 1510 =I 898
1.16 (1.32)
0.92 (1.00)
2.21 (3.33)
0.95 0.67 0.63 0.87
(1.04) (0.72) (0.72) (0.96)
0.83 0.49 0.71 0.60
(0.87) (0.55) (0.77) (0.65)
1.71 1.23 1.38 1.41
(2.61) (1.56) (2.05) (2.12)
0.84 0.82 0.56 0.39
(0.95) (0.93) (0.59) (0.47)
0.78 0.94 0.53 0.50
(0.83) (0.99) (0.54) (0.54)
1.61 1.33 0.82 0.53
(2.32) (1.86) (1.09) (0.75)
Relative intensities were calculated using peak heights (out the parentheses) and areas (in the parentheses).
early stage of pretreatment and the selectivity turned into a non-selective degradation with increasing pretreatment time, which were also found in Pinus radiata decayed by Ganoderma australe (Ferraz et al., 2000). 3.3. Enzymatic hydrolysis of bamboo culms To evaluate the effect of pretreatment with T. versicolor G20 and E. taxodii 2538 on enzymatic hydrolysis of
bamboo culms, we determined fermentable sugar yields of bamboo culms non-pretreated and pretreated after 6, 12, 24, 48, 72, 96, 120-h enzymatic hydrolysis. As expected, bamboo culms without pretreatment were much more resistant to enzymatic hydrolysis with only producing 38 mg fermentable sugar per gram substrate after 120-h hydrolysis (Fig. 2). Higher sugar yields were achieved when the bamboo culms pretreated with white rot fungi were used as the substrate. There was a rapid increase in the fermentable sugar yield with increasing pretreatment time during the early stage of pretreatment. However, the yield has little change after pretreating the bamboo culms with T. versicolor G20 for more than 60 days and with E. taxodii 2538 for more than 90 days. Fermentable sugar yields increased 5.15-fold and 8.76-fold, respectively, after the 120-day pretreatment with T. versicolor G20 and E. taxodii 2538. Although prolonging the biological pretreatment could increase the fermentable sugar yield, the pretreatment cost and feedstock loss would increase, too. For example, T. versicolor G20 caused 40.02% weight loss after the 120-day pretreatment. Therefore, it is important to make the balance of the pretreatment time and the increase of the fermentable sugar yield for biological pretreatment of bamboo culms. E. taxodii 2538 improved the enzymatic hydrolysis of bamboo culms more rapidly than T. versicolor G20. The fermentable sugar yield of bamboo culms pretreated with E. taxodii 2538 for 60 days was similar to that with T. versicolor G20 for 120 days, while E. taxodii 2538 caused lower weight loss (26.31%) than T. versicolor G20. Thus, E. taxodii 2538 was the more
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promising fungus for biological pretreatment than T. versicolor G20.
significant increase of the initial adsorption capacity and decrease of the lignin content with increasing pretreatment time. The initial adsorption capacity of bamboo culms pretreated with E. taxodii 2538 was higher than with T. versicolor G20, while lignin content of that was lower because E. taxodii 2538 is a selectivity lignin-degrading fungus. The initial adsorption capacity increased 6.66-fold and lignin content decreased 29.14% after 120-day pretreatment with E. taxodii 2538, while the initial adsorption capacity increased 4.20-fold and lignin content decreased 12.00% with T. versicolor G20. Data on the initial adsorption capacity, lignin content and fermentable sugar yield of bamboo non-pretreated and pretreated with two fungi for different days were analyzed by linear regression in order to further understanding the effect of adsorption capacity and lignin content on the hydrolysis. Fig. 3 showed the relationship between the initial adsorption capacity, lignin content and enzymatic hydrolysis. The adsorption of the cellulase to the lignocellulose is the first step in the hydrolysis reaction. Previous studies reported the low adsorption capacity resulted in the lack of accessibility of the enzymes to the lignocellulose
3.4. Effect of initial adsorption capacity and lignin content on enzymatic hydrolysis The increase of the fermentable sugar yield of pretreated bamboo culms indicated the decrease of the recalcitrance to enzymatic hydrolysis. The changes of the recalcitrance resulted from the altering of the bamboo characteristic during the pretreatment with white rot fungi. These characteristics may include adsorption capacity to cellulase and lignin content (Saddler, 1999; Gardner et al., 1999; Mooney et al., 1998; Sun and Cheng, 2002). Table 3 showed the initial adsorption capacity to cellulase and lignin content of bamboo culms after pretreatment with white rot fungi. The pretreated bamboo culms showed 400 Fermentable sugar yield (mg·g-1)
163
350 300 250
Table 3 Initial adsorption capacity to cellulase and lignin content of the Moso bamboo culms pretreated with Trametes versicolor G20 and Echinodontium taxodii 2538
200 150
Pretreatment Initial adsorption capacity to Lignin content (%) time (days) cellulase (%)
100 50
Trametes versicolor G20
0 0
20
40 60 80 100 Hydrolysis time (hours)
120 0 30 60 90 120
Fig. 2. Time course of fermentable sugar yield (mg g1 substrate) during the hydrolysis of moso bamboo culms non-pretreated ð&Þ and pretreated with Trametes versicolor G20 (—) and Echinodontium taxodii 2538 ð. . .Þ for 30 ð’Þ, 60 ðÞ, 90 ðmÞ, 120 ð.Þ days with 20 FPU g1 enzyme loading.
4.96 (0.35) (0.50) 25.66 (2.52) (0.44) 29.86 (3.15) (3.55) 43.78 (2.65) (2.02) 37.98 (2.65)
0.179 0.158 0.164 0.154
Echinodontium taxodii 2538
0.175 (0.005) (0.007) 0.169 (0.006) (0.000) 0.157 (0.001) (0.011) 0.121 (0.010) (0.014) 0.124 (0.007)
400
350
R2=0.817
300 250 200 150 100
R2=0.918
50
Fermentable sugar yield (mg·g-1)
Fermentable sugar yield (mg·g-1)
400
20.31 20.62 21.35 25.84
Echinodontium Trametes taxodii 2538 versicolor G20
350 300
R2=0.937
250 200 150
R2=0.829
100 50 0
0 0
10 20 30 40 50 Intial adsorption capacity to cellulase (%)
0.10
0.12
0.14 0.16 Lignin content (%)
0.18
0.20
Fig. 3. Effects of initial adsorption capacity to cellulase (A) and lignin content (B) on enzymatic hydrolysis of bamboo culms. Solid circles: fermentable sugar yield ðmg g1 ) after 6-h enzymatic hydrolysis. Hollow circles: fermentable sugar yield ðmg g1 ) after 120-h enzymatic hydrolysis.
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X. Zhang et al. / International Biodeterioration & Biodegradation 60 (2007) 159–164
(Saddler, 1999; Sun and Cheng, 2002). Therefore, adsorption capacity is a key factor that affects enzymatic hydrolysis. As expected, early fermentable sugar yield (6 h) was positively related to the initial adsorption capacity. However, sugar yields became more uncorrelated to the initial adsorption capacity with increasing hydrolysis time and R2 value of 120 h-hydrolysis was 0.817. Some studies indicated that lignin could influence the release of the cellulase adsorbed on the residual substrate into the reaction filtrate and impede the cellulase recycle (Gregg and Saddler, 1996; Eriksson et al., 2002). So the final sugar yield was also influenced by lignin content besides the adsorption capacity. In fact, Fig. 3B showed that the final fermentable sugar yield (120 h) was negatively related to the lignin content significantly (R2 ¼ 0:937) by the linear regression analysis in spite of non-pretreated bamboo culms. However, the sharp increases of the final fermentable sugar yield during the early pretreatment (30-day) cannot be explained by the decrease in the content of lignin. The short perpendicular lines in Fig. 2 showed the hydrolysis yield of bamboo culms increased sharply after 30-day pretreatment and the lignin content had little change. So the increase of hydrolysis yield may be mainly attributed to the sharp increase of adsorption capacity during the early pretreatment (Table 3). In conclusion, biological pretreatment with white rot fungi significantly improved the enzymatic hydrolysis of bamboo culms by decreasing the lignin content and increasing the initial adsorption capacity. E. taxodii 2538, a selective lignindegrading fungus, improved the hydrolysis more significantly than T. versicolor G20 because bamboo culms pretreated with it had lower lignin content and higher initial adsorption capacity. Acknowledgments The authors thank the support from the Opening Foundation of State Key Laboratory of Pulp and Paper Engineering in South China University of Technology (200439). References Behera, B.K., Arora, M., Sharma, D.K., 1996. Scanning electron microscopic (SEM) studies on structural architecture of lignocellulosic materials of calotropis procera during its processing for saccharification. Bioresource Technology 58, 241–245. Eriksson, T., Bo¨rjesson, J., Tjerneld, F., 2002. Mechanism of surfactant effect in enzymatic hydrolysis of lignocellulose. Enzyme and Microbial Technology 31, 353–364. Ferraz, A., Parra, C., Freer, J., Baeza, J., Rodrı´ guez, J., 2000. Characterization of white zones produced on Pinus radiata wood chips by Ganoderma australe and Ceriporiopsis subvermispora. World Journal of Microbiology and Biotechnology 16, 641–645.
Gardner, T.P., Wood, T.J., Chesson, A., Stuchbury, T., 1999. Effect of degradation on the porosity and surface area of forage cell walls of differing lignin content. Journal of the Science of Food and Agriculture 79, 11–18. Gregg, D.J., Saddler, J.N., 1996. Factors affecting cellulose hydrolysis and the potential of enzyme recycle to enhance the efficiency of an integrated wood to ethanol process. Biotechnology and Bioengineering 51, 375–383. Hakalaa, T.K., Maijala, P., Konnb, J., Hatakkaa, A., 2004. Evaluation of novel wood-rotting polypores and corticioid fungi for the decay and biopulping of Norway spruce (Picea abies) wood. Enzyme and Microbial Technology 34, 255–263. Hakalaa, T.K., Lundella, T., Galkina, S., Maijalaa, P., Kalkkinenb, N., Hatakkaa, A., 2005. Manganese peroxidases, laccases and oxalic acid from the selective white-rot fungus Physisporinus rivulosus grown on spruce wood chips. Enzyme and Microbial Technology 36, 461–468. Horwitz, W. (Ed.), 1980. Official Methods of Analysis of the Association of Official Analytical Chemists. Association of Official Analytical Chemists, Washington USA. Itoh, H., Wada, M., Honda, Y., Kuwahara, M., Watanabe, T., 2003. Bioorganosolve pretreatments for simultaneous saccharification and fermentation of beech wood by ethanolysis and white rot fungi. Journal of Biotechnology 103, 273–280. Kobayashi, F., Take, H., Asada, C., Nakamura, Y., 2004. Methane production from steam-exploded bamboo. Journal of Bioscience and Bioengineering 97, 426–428. Lee, A.W.C., Chen, G., Tainter, F.H., 2001. Comparative treatability of Moso bamboo and Southern pine with CCA preservative using a commercial schedule. Bioresource Technology 77, 87–88. Mata, G., Savoie, J.M., 1998. Extracellular enzyme activities in six Lentinula edodes strains during cultivation in wheat straw. World Journal of Microbiology and Biotechnology 14, 513–519. Mooney, C.A., Mansfield, S.D., Touhy, M.G., Saddler, J.N., 1998. The effect of initial pore volume and lignin content on the enzymatic hydrolysis of softwoods. Bioresource Technology 64, 113–119. Mooney, C.A., Mansfield, S.D., Beatson, R.P., Saddler, J.N., 1999. The effect of fiber characteristics on hydrolysis and cellulase accessibility to softwood substrates. Enzyme and Microbial Technology 25, 644–650. Pandeya, K.K., Pitmanb, A.J., 2003. FTIR studies of the changes in wood chemistry flowing decay by brown-rot and white-rot fungi. International Biodeterioration & Biodegradation 52, 151–160. Rezaeian, M., Beakes, G.W., Chaudhry, A.S., 2005. Relative fibrolytic activities of anaerobic rumen fungi on untreated and sodium hydroxide treated barley straw in vitro culture. Anaerobe 11, 163–175. Saddler, J.N., 1999. Adsorption and activity profiles of cellulases during the hydrolysis of two Douglas fir pulps. Enzyme and Microbial Technology 24, 138–143. Scurlock, J.M.O., Dayton, D.C., Hames, B., 2000. Bamboo: an overlooked biomass resource? Biomass and Bioenergy 19, 229–244. Sun, Y., Cheng, J., 2002. Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresource Technology 83, 1–11. Taniguchi, M., Suzuki, H., Watanabe, D., Sakai, K., Hoshino, K., Tanaka, T., 2005. Evaluation of pretreatment with Pleurotus ostreatus for enzymatic hydrolysis of rice straw. Journal of Bioscience and Bioengineering 100, 637–643. Tsuda, M., Aoyama, M., Cho, N.S., 1998. Catalyzed steaming as pretreatment for the enzymatic hydrolysis of bamboo grass culms. Bioresource Technology 64, 241–243. Vu, T.H.M., Pakkanen, H., Ale´n, R., 2004. Delignification of bamboo (Bambusa procera acher)—Part 1. Kraft pulping and the subsequent oxygen delignification to pulp with a low kappa number. Industrial Crops and Products 19, 49–57.
International Biodeterioration & Biodegradation 60 (2007) 159–164 www.elsevier.com/locate/ibiod
Evaluation of biological pretreatment with white rot fungi for the enzymatic hydrolysis of bamboo culms Xiaoyu Zhang, Hongbo Yu, Huiyan Huang, Youxun Liu College of Life Science and Technology, Huazhong University of Science and Technology, 430074 Wuhan, P.R. China Received 27 November 2006; received in revised form 9 February 2007; accepted 9 February 2007 Available online 25 April 2007
Abstract Biological pretreatment with white rot fungi has shown potential for improving enzymatic hydrolysis of wood and grass. In this study, 34 isolates of white rot fungi were screened for the biological pretreatment of bamboo culms (Phyllostachys pubescence). Echinodontium taxodii 2538 and Trametes versicolor G20 were selected for further evaluation of pretreatment because they caused high lignin loss ð420%Þ and high selectivity value of lignin degradation ð42Þ after the 4-week biodegradation. Fermentable sugar yield of bamboo culms pretreated with these two fungi through enzymatic hydrolysis increased with increasing pretreatment time. Sugar yield of bamboo culms pretreated with T. versicolor G20 and E. taxodii 2538 increased 5.15-fold and 8.76-fold, respectively, after 120-day pretreatment. FTIR analysis showed that E. taxodii 2538 preferentially degraded the lignin of bamboo culms. The pretreated bamboo culms showed significant increase of initial adsorption capacity to cellulase (4.20-fold and 6.66-fold for T. versicolor G20 and E. taxodii 2538, respectively, after 120 days) and decrease of lignin content (12.00% and 29.14% for T. versicolor G20 and E. taxodii 2538, respectively, after 120 days) with increasing pretreatment time. Initial adsorption capacity and lignin content of bamboo culms were correlated to fermentable sugar yield. Scientific relevance This paper focused on the biodegradation and pretreatment of bamboo culms with white rot fungi. Bamboo culms pretreatment with white rot fungi was evaluated firstly for energy convention of lignocellulose. This paper studied effects of lignin content and initial adsorption capacity on enzymatic hydrolysis also. r 2007 Elsevier Ltd. All rights reserved. Keywords: Trametes versicolor; Echinodontium taxodii; Bamboo culms; Biological pretreatment; Enzymatic hydrolysis; Lignin; Adsorption
1. Introduction Bamboos are perennial woody grasses belonging to the Gramineae family and Bambuseae subfamily and distribute widely in Asia (Lee et al., 2001; Scurlock et al., 2000; Vu et al., 2004). China is a major bamboo-producing country and has about 300 species in 44 genera, occupying 3% of total forest area (Scurlock et al., 2000). Bamboo has been widely used as the feedstock of paper, textile, food, construction and reinforcing fibers. Recently, some studies indicated bamboo culms could be exploited as the feedstock of biomass energy, such as ethanol and methanol (Tsuda et al., 1998; Kobayashi et al., 2004; Scurlock et al., 2000). Corresponding author. Tel.: +86 27 8779 2128; fax: +86 27 8779 2108.
E-mail address: [email protected] (X. Zhang). 0964-8305/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2007.02.003
It is necessary to degrade the lignin by the pretreatment for energy production from bamboo culms since the lignin network prevents enzymatic treatment of bamboo culms and release of fermentable sugars. White rot fungi are the only known organisms efficiently removing lignin from the plant cell walls in the world (Hakalaa et al., 2005). Biological pretreatment of lignocellulose with white rot fungi is a promising technology for energy production from lignocellulose because of the advantages of low energy requirement and mild environmental conditions (Hakalaa et al., 2004; Sun and Cheng, 2002). Some studies indicated the pretreatment with white rot fungi could reduce lignocellulose recalcitrance to enzymatic hydrolysis and improve the technological process of energy production from wood and grass (Itoh et al., 2003; Taniguchi et al., 2005). Currently, some physical and chemical pretreatment
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have been used for the enzymatic hydrolysis of bamboo culms in the energy production (Tsuda et al., 1998; Kobayashi et al., 2004). However, biological pretreatment of bamboo culms with white rot fungi had been neglected. Bamboo culms have differences from wood and grass in chemical constituents and fiber characteristic (Scurlock et al., 2000). Accordingly, it is necessary to evaluate the pretreatment with white rot fungi for enzymatic hydrolysis of bamboo culms. In this study, 34 isolates of white rot fungi were screened for the biological pretreatment of Moso bamboo culms (Phyllostachys pubescence) by the ability to degrade bamboo lignin and selectivity value of lignin degradation. Pretreatments of bamboo culms with Echinodontium taxodii 2538 and Trametes versicolor G20 which were selected according to the results of the first screening step were evaluated in more details including saccharification, lignin content and initial adsorption capacity. Biodegradation patterns of the pretreated bamboo culms were also characterized by FTIR analysis.
2. Materials and methods 2.1. Microorganisms and isolation Most of the fungi used in the evaluation were isolated from basidiocarps of white rot fungi which were collected in Shenlongjia nature reserve (Hubei, China) and caused great decay of host plant. Other fungi were obtained from our laboratory. Isolates were recovered by placing small pieces of fruit body plectenchyma aseptically or decayed wood under the fruit body on potato dextrose agar (PDA) containing streptomycin ð50 mg l1 Þ to prevent growth of bacteria. Stock cultures of fungi were maintained on PDA slants at 4 1C. The species were listed in Table 1. Fungi from Shenlongjia nature reserve were identified based on their macro and microscopic morphological characteristics by Dr. Yucheng Dai and Dr. Kari T. Steffen. E. taxodii 2538 were further identified by comparing the nucleotide sequences of 5.8S rRNA gene with those of related strains in GenBank. The GenBank accession number of E. taxodii 2538 was EF422215.
Table 1 Percent losses of different components of Moso bamboo (Phyllostachys pubescence) culms after 4-week pretreatment by 34 white rot fungi Species and strainsa
Antrodiella zonata 5893 Boletus sp. P44b Collybia sp. 2813 Daedaleopsis confrgosa 246 Echinodontium taxodii 2538d Flammulina velutipes WFHb Fomes fomentarius 248 Ganoderma australe P17c
Selectivity Component loss (%) value Weight Lignin Cellulose Hemicellulose
Table 1 (continued ) Species and strainsa
Selectivity Component loss (%) value Weight Lignin Cellulose Hemicellulose
Ganoderma lipsisense 245 Ganoderma lucidum En4 Ganoderma sp. En1b Ganoderma sp. En3 Ganoderma sp. En5 Grifola umbellata P64b Hygrocybe punicea 258 Pachykytospora papyracea 5865 Perenniporia sp. 5814 Pleurotus ostreatus Bp2b Polyporus mongolius 2410 Rigidoporus crocatus 5899 Russula sp. 2416 Schizopora flavipora 5951 Trametes gibbosa 5955 Trametes ochracea 243 Trametes ochracea 5930 Trametes ochracea 5897 Trametes ochracea P58 Trametes versicolor CD1 Trametes versicolor G20 Trametes sp. Yhb-5 Trichaptum abietinum 247 Trichaptum biforme 2526 Trichaptum sp. P39 Tyromyces chioneus 5877
2.74
10.78
16.25 5.92
19.31
0.82
12.10
10.56 12.83
15.16
1.15 2.30 0.58 1.11
6.76 8.36 2.59 8.26
5.10 15.72 1.72 10.01
4.45 6.84 2.99 9.00
17.30 9.95 0.05 13.85
1.68
3.13
6.59
3.93
0.60
None
None
None None
None
6.61 0.61
2.51 5.17
8.66 2.67
1.31 4.38
3.36 12.59
3.88
9.01
13.07 3.37
16.13
None
None
None None
None
None None
None None
None None None None
None None
1.45
11.72
13.40 9.22
24.77
0.86
15.49
13.26 15.49
27.24
2.90
11.05
18.20 6.27
21.88
0.54
10.23
5.50
10.23
25.06
1.73
15.21
18.63 10.79
29.22
4.25
6.62
9.14
1.75
2.61
13.63
24.42 9.35
18.89
2.12 1.25
7.37 10.14
13.16 6.22 13.56 10.86
9.78 21.08
1.48
11.04
12.54 8.48
32.70
0.86 1.85
9.19 3.18
7.60 1.44
25.78 13.29
2.15
8.81 0.78
a
Fungi from shenlongjia nature reserve were identified by Dr. Yucheng Dai and Dr. Kari T. Steffen. b Obtained from our laboratory. c Obtained from YiChang, Hubei province, and identified by Dr. Yucheng Dai. d The GenBank accession number: EF422215.
1.52
6.34
5.50
3.63
17.74
6.00 0.64 1.65
2.85 11.28 8.74
0.24 8.12 8.74
0.04 12.75 5.30
12.14 20.54 28.58
14.8
10.58
24.28 1.64
28.46
2.2. The first screening step
0.81
2.27
3.14
3.88
4.82
6.24
0.50
3.12
0.50
0.50
1.23
9.11
7.49
6.11
18.60
Moso bamboo (Phyllostachys pubescence) culms from Wuhan were ground to pass through a 0.9 mm screen and then were kiln-dried at 60 1C for three days. In order to select the best strains for the subsequent pretreatment evaluation, 34 fungi were screened by lignin-degrading ability and selectivity value of lignin degradation. The experiments were carried out in Petri dishes with 5 g bamboo culms and 10 ml of synthetic
ARTICLE IN PRESS X. Zhang et al. / International Biodeterioration & Biodegradation 60 (2007) 159–164 medium with the following composition per liter: MgCl2 0.01 g, KH2PO4 0.002 g, CaCl2 0.0005 g and yeastrel 0.05 g. Petri dishes were used as the decay chambers. Film was wrapped around Petri dishes to act as a barrier against moisture loss and contamination. Small perforations were made to the film to avoid moisture condensation and allow ventilation of chambers. All isolates of white rot fungi were grown on PDA plate at 25 1C for 10 days before cutting inoculating plugs. Petri dishes were sterilized in the autoclave for 20 min at 121 1C and aseptically inoculated with a plug cut from the margins of the PDA culture. After Petri dishes were incubated at 25 1C for four weeks, the mycelium covering the bamboo in Petri dishes was gently removed. The decayed bamboo culms were dried at 60 1C for three days and then weighted to calculate weight losses based on the initial and final dry weights. Lignin and cellulose were determined according to procedures of AOAC (1980) (Horwirtz, 1980). Hemicellulose was determined by the difference between neutral detergent fiber (NDF) and acid detergent fiber (ADF) (Rezaeian et al., 2005). The selectivity value of lignin degradation was calculated as lignin loss/ cellulose loss ratio.
2.3. Bamboo culms pretreatment with white rot fungi The biological pretreatments with E. taxodii 2538 and T. versicolor G20 were carried out in 250-ml Erlenmeyer flasks with 10 g ground bamboo culms and 20 ml distilled water. Flasks were sterilized in the autoclave for 20 min at 121 1C and aseptically inoculated with a plug cut from the margins of the PDA culture. Cultures were maintained statically at 25 1C for 30, 60, 90 and 120 days and then dried at 60 1C for three days removing mycelium covering the bamboo. All the cultures were grown in triplicate. Non-inoculated bamboo was used as control.
2.4. FTIR analysis of biodegradation patterns Bruker Vertex 70 infrared spectrophotometer was used to investigate biodegradation patterns of bamboo culms pretreated with E. taxodii 2538 and T. versicolor G20. The dried bamboo samples were mixed with KBr of spectroscopic grade and made in the form of pellets at pressure of about 1 MPa. The pellets were about 10 mm in diameter and 1 mm thickness. Peak height and area were determined by Omnic software and relative changes in the intensity of lignin/carbohydrate characteristic bands were analyzed by Pandeya’s method (Pandeya and Pitmanb, 2003).
2.5. Enzymatic hydrolysis Enzymatic hydrolysis experiments of bamboo culms pretreated with E. taxodii 2538 and T. versicolor G20 were performed to investigate effect of biological pretreatment. Hydrolysis was carried out at 2.5% bamboo substrate concentration in 50 mM sodium acetate buffer (pH 4.8) with cellulase (20 FPU g1 substrate) at 50 1C. Cellulase was obtained from Sigma. After 6, 12, 24, 48, 72, 96, 120 h, the release of fermentable sugars was determined with the DNS (3,5-dinitrosalicylic acid) reagent (Behera et al., 1996; Mata and Savoie, 1998).
2.6. Adsorption measurements An enzyme loading of 20 FPU g1 bamboo substrate was used to investigate initial adsorption capacity to cellulase. Adsorption reaction was performed as described by Mooney et al. (1998) in 50 mM sodium acetate buffer (pH 4.8) for 90 min at 4 1C and then for 10 min at 50 1C. Protein in the centrifugate was measured using the Bradford protein assay after centrifugation at 10 000 rpm for 5 min (Mooney et al., 1999). Adsorbed cellulase protein was calculated based on the difference between the amount of initial protein and unadsorbed protein in the centrifugate. Initial adsorption capacity of bamboo substrate was calculated as percent ratio of adsorbed protein/initial protein.
161
3. Results and discussion 3.1. The first screening step The weight and component losses of bamboo culms pretreated by 34 fungi were listed in Table 1. All fungi caused the delignification of bamboo culms except for four fungi which were not able to grow in the bamboo culms. In most cases, white rot fungi caused higher hemicellulose loss than other component losses. Although Trametes ochracea 243 had the greatest degrading ability in all tested fungi after the 4-week of cultivation, it was not suitable for bamboo pretreatment because it simultaneously degraded cellulose and lignin (selectivity valueo1). Low selectivity value meant relatively high cellulose loss during the biological pretreatment. Thus, the selectivity value of lignin degradation and lignin loss were used to screen white rot fungi for biological pretreatment. Eleven strains had great selective lignin-degrading ability (selectivity value42). Among these fungi, E. taxodii 2538 and T. versicolor G20 caused the greatest lignin loss. Therefore, E. taxodii 2538 and T. versicolor G20 were selected to further study for evaluation of bamboo culms pretreatment. 3.2. Evaluation of biodegradation patterns Biodegradation patterns of bamboo culms during the biological pretreatment with E. taxodii 2538 and T. versicolor G20 were evaluated by FTIR analysis. Fig. 1A and B showed the FTIR spectra of bamboo culms pretreated with two fungi for different days. Spectrum of bamboo culms pretreated with E. taxodii 2538 revealed a proportional decrease in intensity of lignin peak at 1510 cm1 which was assigned for aromatic skeletal in lignin with prolonged pretreatment (Pandeya and Pitmanb, 2003). There were little changes at 1510 cm1 peak of bamboo culms pretreated with T. versicolor G20 although these two fungi had similar lignin-degrading ability. In order to fully understand biodegradation patterns of bamboo culms, we made a detailed FTIR spectroscopic analysis based on Pandeya’s analysis method (Pandeya and Pitmanb, 2003). Table 2 showed relative changes in the intensities of lignin peaks at 1510 cm1 against carbohydrate peaks at 1734, 1378, 1163 and 898 cm1 which were calculated by peak heights and areas. These four carbohydrate peaks were assigned, respectively, for unconjugated CQO in xylans, C–H deformation in cellulose and hemicellulose, C–O–C vibration in cellulose and hemicellulose and C–H deformation in cellulose (Pandeya and Pitmanb, 2003). There was significant decrease in the lignin/carbohydrate ratio of bamboo pretreated by E. taxodii 2538 with increasing pretreatment time, which indicated the fungus had great selective lignin-degrading ability. The lignin/carbohydrate ratio of bamboo pretreated with T. versicolor G20 increased at advanced stages of pretreatment although it decreased at the early stage (before 60 days). These results indicated that T. versicolor G20 preferentially degrade lignin only at the
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162
(4) (2)
(1)
(1)
(3)
(4) (5)
e Absorbance
Absorbance
(3)
(5)
e d
d c
c
b
b
a 1800
(2)
a 1600
1400
1200
Wavenumbers
1000
800
1800
1600
1400
1200
1000
800
Wavenumbers (cm-1)
(cm-1)
Fig. 1. FTIR spectra of bamboo culms pretreated with Trametes versicolor G20 (A) and Echinodontium taxodii 2538 (B) for different days: (a) control; (b) pretreated for 30 days; (c) pretreated for 60 days; (d) pretreated for 90 days; (e) pretreated for 120 days. (1) 1734 cm1 , (2) 1510 cm1 , (3) 1378 cm1 , (4) 1163 cm1 , (5) 898 cm1 .
Table 2 Ratios of the intensity of the lignin associated band with carbohydrate bands for pretreated samples Pretreatment Relative intensities of aromatic skeletal vibration (I 1510 ) against typical bands for carbohydrates time (days) I 1510 =I 1734 Control 0 0.80 (0.57) Trametes versicolor G20 30 0.64 (0.44) 60 0.33 (0.20) 90 0.45 (0.27) 120 0.70 (0.52) Echinodontium taxodii 2538 30 0.53 (0.37) 60 0.48 (0.31) 90 0.38 (0.25) 120 0.26 (0.17)
I 1510 =I 1378
I 1510 =I 1163
I 1510 =I 898
1.16 (1.32)
0.92 (1.00)
2.21 (3.33)
0.95 0.67 0.63 0.87
(1.04) (0.72) (0.72) (0.96)
0.83 0.49 0.71 0.60
(0.87) (0.55) (0.77) (0.65)
1.71 1.23 1.38 1.41
(2.61) (1.56) (2.05) (2.12)
0.84 0.82 0.56 0.39
(0.95) (0.93) (0.59) (0.47)
0.78 0.94 0.53 0.50
(0.83) (0.99) (0.54) (0.54)
1.61 1.33 0.82 0.53
(2.32) (1.86) (1.09) (0.75)
Relative intensities were calculated using peak heights (out the parentheses) and areas (in the parentheses).
early stage of pretreatment and the selectivity turned into a non-selective degradation with increasing pretreatment time, which were also found in Pinus radiata decayed by Ganoderma australe (Ferraz et al., 2000). 3.3. Enzymatic hydrolysis of bamboo culms To evaluate the effect of pretreatment with T. versicolor G20 and E. taxodii 2538 on enzymatic hydrolysis of
bamboo culms, we determined fermentable sugar yields of bamboo culms non-pretreated and pretreated after 6, 12, 24, 48, 72, 96, 120-h enzymatic hydrolysis. As expected, bamboo culms without pretreatment were much more resistant to enzymatic hydrolysis with only producing 38 mg fermentable sugar per gram substrate after 120-h hydrolysis (Fig. 2). Higher sugar yields were achieved when the bamboo culms pretreated with white rot fungi were used as the substrate. There was a rapid increase in the fermentable sugar yield with increasing pretreatment time during the early stage of pretreatment. However, the yield has little change after pretreating the bamboo culms with T. versicolor G20 for more than 60 days and with E. taxodii 2538 for more than 90 days. Fermentable sugar yields increased 5.15-fold and 8.76-fold, respectively, after the 120-day pretreatment with T. versicolor G20 and E. taxodii 2538. Although prolonging the biological pretreatment could increase the fermentable sugar yield, the pretreatment cost and feedstock loss would increase, too. For example, T. versicolor G20 caused 40.02% weight loss after the 120-day pretreatment. Therefore, it is important to make the balance of the pretreatment time and the increase of the fermentable sugar yield for biological pretreatment of bamboo culms. E. taxodii 2538 improved the enzymatic hydrolysis of bamboo culms more rapidly than T. versicolor G20. The fermentable sugar yield of bamboo culms pretreated with E. taxodii 2538 for 60 days was similar to that with T. versicolor G20 for 120 days, while E. taxodii 2538 caused lower weight loss (26.31%) than T. versicolor G20. Thus, E. taxodii 2538 was the more
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promising fungus for biological pretreatment than T. versicolor G20.
significant increase of the initial adsorption capacity and decrease of the lignin content with increasing pretreatment time. The initial adsorption capacity of bamboo culms pretreated with E. taxodii 2538 was higher than with T. versicolor G20, while lignin content of that was lower because E. taxodii 2538 is a selectivity lignin-degrading fungus. The initial adsorption capacity increased 6.66-fold and lignin content decreased 29.14% after 120-day pretreatment with E. taxodii 2538, while the initial adsorption capacity increased 4.20-fold and lignin content decreased 12.00% with T. versicolor G20. Data on the initial adsorption capacity, lignin content and fermentable sugar yield of bamboo non-pretreated and pretreated with two fungi for different days were analyzed by linear regression in order to further understanding the effect of adsorption capacity and lignin content on the hydrolysis. Fig. 3 showed the relationship between the initial adsorption capacity, lignin content and enzymatic hydrolysis. The adsorption of the cellulase to the lignocellulose is the first step in the hydrolysis reaction. Previous studies reported the low adsorption capacity resulted in the lack of accessibility of the enzymes to the lignocellulose
3.4. Effect of initial adsorption capacity and lignin content on enzymatic hydrolysis The increase of the fermentable sugar yield of pretreated bamboo culms indicated the decrease of the recalcitrance to enzymatic hydrolysis. The changes of the recalcitrance resulted from the altering of the bamboo characteristic during the pretreatment with white rot fungi. These characteristics may include adsorption capacity to cellulase and lignin content (Saddler, 1999; Gardner et al., 1999; Mooney et al., 1998; Sun and Cheng, 2002). Table 3 showed the initial adsorption capacity to cellulase and lignin content of bamboo culms after pretreatment with white rot fungi. The pretreated bamboo culms showed 400 Fermentable sugar yield (mg·g-1)
163
350 300 250
Table 3 Initial adsorption capacity to cellulase and lignin content of the Moso bamboo culms pretreated with Trametes versicolor G20 and Echinodontium taxodii 2538
200 150
Pretreatment Initial adsorption capacity to Lignin content (%) time (days) cellulase (%)
100 50
Trametes versicolor G20
0 0
20
40 60 80 100 Hydrolysis time (hours)
120 0 30 60 90 120
Fig. 2. Time course of fermentable sugar yield (mg g1 substrate) during the hydrolysis of moso bamboo culms non-pretreated ð&Þ and pretreated with Trametes versicolor G20 (—) and Echinodontium taxodii 2538 ð. . .Þ for 30 ð’Þ, 60 ðÞ, 90 ðmÞ, 120 ð.Þ days with 20 FPU g1 enzyme loading.
4.96 (0.35) (0.50) 25.66 (2.52) (0.44) 29.86 (3.15) (3.55) 43.78 (2.65) (2.02) 37.98 (2.65)
0.179 0.158 0.164 0.154
Echinodontium taxodii 2538
0.175 (0.005) (0.007) 0.169 (0.006) (0.000) 0.157 (0.001) (0.011) 0.121 (0.010) (0.014) 0.124 (0.007)
400
350
R2=0.817
300 250 200 150 100
R2=0.918
50
Fermentable sugar yield (mg·g-1)
Fermentable sugar yield (mg·g-1)
400
20.31 20.62 21.35 25.84
Echinodontium Trametes taxodii 2538 versicolor G20
350 300
R2=0.937
250 200 150
R2=0.829
100 50 0
0 0
10 20 30 40 50 Intial adsorption capacity to cellulase (%)
0.10
0.12
0.14 0.16 Lignin content (%)
0.18
0.20
Fig. 3. Effects of initial adsorption capacity to cellulase (A) and lignin content (B) on enzymatic hydrolysis of bamboo culms. Solid circles: fermentable sugar yield ðmg g1 ) after 6-h enzymatic hydrolysis. Hollow circles: fermentable sugar yield ðmg g1 ) after 120-h enzymatic hydrolysis.
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(Saddler, 1999; Sun and Cheng, 2002). Therefore, adsorption capacity is a key factor that affects enzymatic hydrolysis. As expected, early fermentable sugar yield (6 h) was positively related to the initial adsorption capacity. However, sugar yields became more uncorrelated to the initial adsorption capacity with increasing hydrolysis time and R2 value of 120 h-hydrolysis was 0.817. Some studies indicated that lignin could influence the release of the cellulase adsorbed on the residual substrate into the reaction filtrate and impede the cellulase recycle (Gregg and Saddler, 1996; Eriksson et al., 2002). So the final sugar yield was also influenced by lignin content besides the adsorption capacity. In fact, Fig. 3B showed that the final fermentable sugar yield (120 h) was negatively related to the lignin content significantly (R2 ¼ 0:937) by the linear regression analysis in spite of non-pretreated bamboo culms. However, the sharp increases of the final fermentable sugar yield during the early pretreatment (30-day) cannot be explained by the decrease in the content of lignin. The short perpendicular lines in Fig. 2 showed the hydrolysis yield of bamboo culms increased sharply after 30-day pretreatment and the lignin content had little change. So the increase of hydrolysis yield may be mainly attributed to the sharp increase of adsorption capacity during the early pretreatment (Table 3). In conclusion, biological pretreatment with white rot fungi significantly improved the enzymatic hydrolysis of bamboo culms by decreasing the lignin content and increasing the initial adsorption capacity. E. taxodii 2538, a selective lignindegrading fungus, improved the hydrolysis more significantly than T. versicolor G20 because bamboo culms pretreated with it had lower lignin content and higher initial adsorption capacity. Acknowledgments The authors thank the support from the Opening Foundation of State Key Laboratory of Pulp and Paper Engineering in South China University of Technology (200439). References Behera, B.K., Arora, M., Sharma, D.K., 1996. Scanning electron microscopic (SEM) studies on structural architecture of lignocellulosic materials of calotropis procera during its processing for saccharification. Bioresource Technology 58, 241–245. Eriksson, T., Bo¨rjesson, J., Tjerneld, F., 2002. Mechanism of surfactant effect in enzymatic hydrolysis of lignocellulose. Enzyme and Microbial Technology 31, 353–364. Ferraz, A., Parra, C., Freer, J., Baeza, J., Rodrı´ guez, J., 2000. Characterization of white zones produced on Pinus radiata wood chips by Ganoderma australe and Ceriporiopsis subvermispora. World Journal of Microbiology and Biotechnology 16, 641–645.
Gardner, T.P., Wood, T.J., Chesson, A., Stuchbury, T., 1999. Effect of degradation on the porosity and surface area of forage cell walls of differing lignin content. Journal of the Science of Food and Agriculture 79, 11–18. Gregg, D.J., Saddler, J.N., 1996. Factors affecting cellulose hydrolysis and the potential of enzyme recycle to enhance the efficiency of an integrated wood to ethanol process. Biotechnology and Bioengineering 51, 375–383. Hakalaa, T.K., Maijala, P., Konnb, J., Hatakkaa, A., 2004. Evaluation of novel wood-rotting polypores and corticioid fungi for the decay and biopulping of Norway spruce (Picea abies) wood. Enzyme and Microbial Technology 34, 255–263. Hakalaa, T.K., Lundella, T., Galkina, S., Maijalaa, P., Kalkkinenb, N., Hatakkaa, A., 2005. Manganese peroxidases, laccases and oxalic acid from the selective white-rot fungus Physisporinus rivulosus grown on spruce wood chips. Enzyme and Microbial Technology 36, 461–468. Horwitz, W. (Ed.), 1980. Official Methods of Analysis of the Association of Official Analytical Chemists. Association of Official Analytical Chemists, Washington USA. Itoh, H., Wada, M., Honda, Y., Kuwahara, M., Watanabe, T., 2003. Bioorganosolve pretreatments for simultaneous saccharification and fermentation of beech wood by ethanolysis and white rot fungi. Journal of Biotechnology 103, 273–280. Kobayashi, F., Take, H., Asada, C., Nakamura, Y., 2004. Methane production from steam-exploded bamboo. Journal of Bioscience and Bioengineering 97, 426–428. Lee, A.W.C., Chen, G., Tainter, F.H., 2001. Comparative treatability of Moso bamboo and Southern pine with CCA preservative using a commercial schedule. Bioresource Technology 77, 87–88. Mata, G., Savoie, J.M., 1998. Extracellular enzyme activities in six Lentinula edodes strains during cultivation in wheat straw. World Journal of Microbiology and Biotechnology 14, 513–519. Mooney, C.A., Mansfield, S.D., Touhy, M.G., Saddler, J.N., 1998. The effect of initial pore volume and lignin content on the enzymatic hydrolysis of softwoods. Bioresource Technology 64, 113–119. Mooney, C.A., Mansfield, S.D., Beatson, R.P., Saddler, J.N., 1999. The effect of fiber characteristics on hydrolysis and cellulase accessibility to softwood substrates. Enzyme and Microbial Technology 25, 644–650. Pandeya, K.K., Pitmanb, A.J., 2003. FTIR studies of the changes in wood chemistry flowing decay by brown-rot and white-rot fungi. International Biodeterioration & Biodegradation 52, 151–160. Rezaeian, M., Beakes, G.W., Chaudhry, A.S., 2005. Relative fibrolytic activities of anaerobic rumen fungi on untreated and sodium hydroxide treated barley straw in vitro culture. Anaerobe 11, 163–175. Saddler, J.N., 1999. Adsorption and activity profiles of cellulases during the hydrolysis of two Douglas fir pulps. Enzyme and Microbial Technology 24, 138–143. Scurlock, J.M.O., Dayton, D.C., Hames, B., 2000. Bamboo: an overlooked biomass resource? Biomass and Bioenergy 19, 229–244. Sun, Y., Cheng, J., 2002. Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresource Technology 83, 1–11. Taniguchi, M., Suzuki, H., Watanabe, D., Sakai, K., Hoshino, K., Tanaka, T., 2005. Evaluation of pretreatment with Pleurotus ostreatus for enzymatic hydrolysis of rice straw. Journal of Bioscience and Bioengineering 100, 637–643. Tsuda, M., Aoyama, M., Cho, N.S., 1998. Catalyzed steaming as pretreatment for the enzymatic hydrolysis of bamboo grass culms. Bioresource Technology 64, 241–243. Vu, T.H.M., Pakkanen, H., Ale´n, R., 2004. Delignification of bamboo (Bambusa procera acher)—Part 1. Kraft pulping and the subsequent oxygen delignification to pulp with a low kappa number. Industrial Crops and Products 19, 49–57.