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The effect of biological pretreatment with the selective white-rot fungus Echinodontium taxodii on enzymatic hydrolysis of softwoods and hardwoods
Bioresource Technology 100 (2009) 5170–5175
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The effect of biological pretreatment with the selective white-rot fungus Echinodontium taxodii on enzymatic hydrolysis of softwoods and hardwoods Hongbo Yu a, Guoning Guo b, Xiaoyu Zhang a,*, Keliang Yan a, Chunyan Xu a a b
College of Life Science and Technology, Huazhong University of Science and Technology, Luoyu Road 1037, Wuhan 430074, China Technology Center of Hubei Tobacco (Group) Co. Ltd., Shisheng Road 22, Wuhan 430051, China
a r t i c l e
i n f o
Article history: Received 7 March 2009 Received in revised form 17 May 2009 Accepted 20 May 2009 Available online 9 July 2009 Keywords: Enzymatic hydrolysis Echinodontium taxodii Biological pretreatment Adsorption Wood
a b s t r a c t Selective white-rot fungi have shown potential for lignocellulose pretreatment. In the study, a new fungal isolate, Echinodontium taxodii 2538, was used in biological pretreatment to enhance the enzymatic hydrolysis of two native woods: Chinese willow (hardwood) and China-fir (softwood). E. taxodii preferentially degraded the lignin during the pretreatment, and the pretreated woods showed significant increases in enzymatic hydrolysis ratios (4.7-fold for hardwood and 6.3-fold for softwood). To better understand effects of biological pretreatment on enzymatic hydrolysis, enzyme–substrate interactions were investigated. It was observed that E. taxodii enhanced initial adsorption of cellulase but which did not always translate to high initial hydrolysis rate. However, the rate of change in hydrolysis rate declined dramatically with decreasing irreversible adsorption of cellulase. Thus, the enhancement of enzymatic hydrolysis was attributed to the decline of irreversible adsorption which may result from partial lignin degradation and alteration in lignin structure after biological pretreatment. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Enzymatic hydrolysis is a key step in ethanol production from low-cost lignocellulosic materials (Pu et al., 2008; Zhao et al., 2008). However, the natural resistance of lignocellulose to hydrolysis prevents the release of sugars (Sun and Cheng, 2002; Yang and Wyman, 2008). Some chemical and physicochemical pretreatment processes, such as acid pretreatment, alkaline pretreatment, steam explosion and ammonia fiber explosion, are used to enhance the enzymatic hydrolysis of lignocellulose, but these processes usually require high temperature and operating pressure (Eggeman and Elander, 2005; Teymouri et al., 2005; Tucker et al., 2003). Recently, some studies indicated biological pretreatment could enhance the enzymatic hydrolysis of lignocellulose and had the advantages of mild conditions and low energy consumption (Amirta et al., 2006; Sun and Cheng, 2002). White-rot fungi which can efficiently degrade lignin are usually used in biological pretreatment of lignocellulose (Hakala et al., 2005). Different white-rot fungi vary greatly in the relative rates at which they degrade lignin and carbohydrates in lignocellulose (Hatakka, 2001). The selective lignin-degrading white-rot fungi are considered good candidates for biological pretreatment (Itoh et al., 2003; Taniguchi et al., 2005). In our previous investigation, a new fungal isolate, Echinodontium taxodii 2538, was found to cause selective degradation of lignin * Corresponding author. Tel.: +86 27 87792108; fax: +86 27 87792128. E-mail address: hongbo.fi[email protected] (X. Zhang). 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.05.049
on bamboo culms, and it could significantly enhance the enzymatic hydrolysis of non-woody lignocellulose, such as bamboo culms and corn straw (Zhang et al., 2007). But it is not yet known whether E. taxodii can degrade selectively lignin in woody lignocellulose (wood) and enhance the enzymatic hydrolysis of wood. It is well known that lignocellulose species can also affect the fungal growth and biodegradation patterns (Blanchette, 1991). Accordingly, it is necessary to investigate the biodegradation pattern of wood (including softwood and hardwood) pretreated by E. taxodii and evaluate the effect of the pretreatment on enzymatic hydrolysis in order to fully exploit the potential of the fungus. In the past, some investigators attributed the enhancement of enzymatic hydrolysis after biological pretreatment to the biodegradation of the lignin seal responsible for preventing the penetration of cellulase, but further study is needed as it is likely that fungal delignification also affects the physical properties of the substrate (Taniguchi et al., 2005; Zhang et al., 2007). Enzymatic hydrolysis of insoluble lignocellulose differs considerably from classical solution kinetics (Gan et al., 2005). For instance, in batch reactions enzymatic hydrolysis of lignocellulose has a phase of rapid initial hydrolysis, and this is followed by a sharp decline in hydrolysis rate (Gan et al., 2003; Movagarnejad et al., 2000). Some studies indicated that the low initial adsorption of cellulase to natural substrates could limit the initial hydrolysis rate, and the decline in hydrolysis rate may result from the irreversible adsorption of cellulase during enzymatic hydrolysis (Boussaid and Saddler, 1999; Desai and Converse, 1997). Therefore, fungal
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Nomenclature X V0 t
the hydrolysis ratio (%) the initial hydrolysis rate of enzymatic hydrolysis (%/h) the enzymatic hydrolysis time (h)
pretreatment is likely to affect the cellulase–substrate interaction by altering substrate properties, leading to the enhancement of enzymatic hydrolysis. In this study, E. taxodii was used in biological pretreatment to enhance the enzymatic hydrolysis of common native hardwood and softwood: Chinese willow (Salix babylonica, hardwood) and China-fir (Cunninghamia lanceolata, softwood). The biodegradation patterns of the pretreated woods were also evaluated by wood component losses and Fourier transform infrared (FTIR) analysis. Moreover, adsorption and desorption of cellulase during enzymatic hydrolysis were investigated to better clarify the cause of the improvement of enzymatic hydrolysis.
k
Protein in the centrifugate was measured using the Bradford protein assay (Mooney et al., 1998). Adsorbed cellulase protein was determined based on the difference between the amounts of added protein and protein in the centrifugate. Adsorption ratio was calculated as follows:
Adsorption ratio ð%Þ ¼
Desorption value ¼
Echinodontium taxodii 2538 was found and isolated in Shennongjia Nature Reserve (Hubei, China) (Zhang et al., 2007). Stock culture of the isolate was maintained on potato dextrose agar (PDA) slants at 4 °C. The inoculum was grown on PDA plate at 25 °C for 10 days.
amount of adsorbed protein 100 amount of added protein
In order to evaluate the irreversible adsorption of cellulase, desorption value was used as an indicator in the study. Desorption value was defined as follows:
2. Methods 2.1. Microorganisms and inoculum
the rate retardation constant during enzymatic hydrolysis
maximum of adsorbed protein during hydrolysis amount of adsorbed protein at the end of hydrolysis amount of added protein
The initial adsorption ratio of cellulase to different woods was also determined at a cellulase loading of 20 FPU/g substrate. The initial adsorption reaction was performed in 50 mM sodium acetate buffer (pH 4.8) for 90 min at 4 °C and then for 10 min at 50 °C (Mooney et al., 1999). Protein in the centrifugate was measured after centrifugation at 10,000 rpm for 5 min. Initial adsorption ratio was calculated as follows:
Initial adsorption ratio ð%Þ ¼ 2.2. Biological pretreatment with E. taxodii Chinese willow (Salix babylonica, hardwood) and China-fir (Cunninghamia lanceolata, softwood) woods from Wuhan were ground to pass through a 0.9 mm screen and then were kiln-dried at 60 °C for three days. The biological pretreatments with E. taxodii were carried out in 125-ml Erlenmeyer flasks with 5 g ground woods and 12.5 ml (for hardwood) or 15 ml (for softwood) distilled water. Flasks were sterilized in the autoclave for 20 min at 121 °C and aseptically inoculated with a plug from the plate culture. Cultures were maintained statically at 25 °C for 30, 60, 90 and 120 days, and then dried at 60 °C for three days for enzymatic hydrolysis and chemical analysis. All the cultures were grown in triplicate. A set of non-pretreated sterilized woods were used as control. 2.3. Enzymatic hydrolysis Enzymatic hydrolysis experiments of woods were carried out at 2.5% substrate concentration in 50 mM sodium acetate buffer (pH 4.8) with cellulase (20 FPU/g substrate) at 50 °C. Cellulase was obtained from Sigma. The reaction mixtures were interrupted for different hydrolysis times by the centrifugation at 10,000 rpm for 5 min. The amounts of reducing sugars and protein in the centrifugate were measured. Each time point was analyzed for reducing sugar and protein at least in triplicate.
amount of initial adsorbed protein 100 amount of added protein
2.5. Chemical analysis of woods Dried woods at 60 °C for three days were weighted to determine weight losses. Neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL) and ash in woods were determined according to procedures of Van Soest (1963) and AOAC (Horwitz, 1980). The amount of hemicellulose, cellulose and Klason lignin in woods were calculated as the difference between ADF and NDF, ADF and ADL, ADL and ash, respectively. The selectivity value of lignin degradation was calculated as lignin loss/cellulose loss ratio. The detail process was described as follows: About 1 g of dried wood and 100 ml of neutral detergent solution with sodium lauryl sulphate (SDS) and disodium ethylene diamine tetra acetate (EDTA) were added into a flat bottom flask for the determination of NDF. The mixture was heated to boiling in 5–10 min and refluxed for 60 min from onset of boiling. The hot mixture was filtered through a previously weighed filtering crucible. The residue was rinsed with a minimum of 400 ml hot distilled water and 10 ml acetone, and then was dried at 105 °C until a constant weight was achieved. NDF ð%Þ ¼
Weight of crucible and residue after treating with neutral detergent solution Weight of crucible 100 Weight of wood
2.4. Determination of reducing sugar and cellulase adsorption
ADF was determined using acid detergent solution with cetyl trimethyl ammonium bromide (CTAB) and 0.5 M H2SO4. The procedure was the same as the determination of NDF.
Reducing sugar was measured with the DNS (3,5-dinitrosalicylic acid) reagent (Behera et al., 1996). The hydrolysis ratio was calculated as follows:
ADF ð%Þ ¼
Weight of crucible and residue after treating with neutral detergent solution Weight of crucible 100 Weight of wood
Hydrolysis ratio ð%Þ ¼
amount of reducing sugar produced after enzymatic hydrolysis 0:9 100 amount of cellulose þ amount of hemicellulose
ADF was digested with 72% H2SO4 for 3 h to determine the ADL. After the digestion, the mixture was filtered through the filtering
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crucible and washed with a minimum of 300 ml hot water until acid-free to pH paper. The residue was dried to a constant weight at 105 °C and represents the ADL. ADL ð%Þ ¼
Weight of crucible and residue after treating with 72% H2 SO4 Weight of crucible 100 Weight of wood
The ash was determined as the residue after treatment in the muffle furnace at 575 °C for 24 h.
Ash ð%Þ ¼
Weight of crucible and ash Weight of crucible 100 Weight of wood
Bruker Vertex 70 infrared spectrophotometer was used FTIR analysis of wood samples. The dried woods 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 of Fourier transform infrared spectra were determined by Omnic software. The relative changes in the intensities of lignin peaks at 1506/ 1510 cm1 (hardwood/softwood) against carbohydrate peaks at 1737/1734 (hardwood/softwood), 1378/1373 (hardwood/softwood), 1161 and 898 cm1 were calculated by peak heights and areas (Pandey and Pitman, 2003). The lignin peaks were assigned for aromatic skeletal in lignin. The four carbohydrate peaks were assigned, respectively, for unconjugated C@O in xylans, C–H deformation in cellulose and hemicellulose, C–O–C vibration in cellulose and hemicellulose and C–H deformation in cellulose. 2.6. Data treatment An empirical expression proposed by Ohmine et al. (1983) was used for data treatment of enzymatic hydrolysis, as Eq. (1) shows:
X¼
1 lnð1 þ V 0 k tÞ k
ð1Þ
where X is the hydrolysis ratio (%), V0 is the initial hydrolysis rate (%/h), t is the hydrolysis time (h) and k is the rate retardation constant. According to Eq. (1), initial hydrolysis rate and the rate of change in the hydrolysis rate which was represented by rate retardation constant were determined by regression analysis of the experimental result. Data fit was performed by software Origin 7.5.
Table 1 Percent losses of different components of the hardwood (Chinese willow) and softwood (China-fir) pretreated with Echinodontium taxodii 2538. Pretreatment time (day)
Selectivity value
Component loss (%) Weight
Lignin
Cellulose
Hemicellulose
Hardwood (Chinese willow) 30 28.9 60 3.0 90 2.0 120 1.7
16.0 ± 0.3 22.1 ± 0.1 30.7 ± 0.7 32.5 ± 1.7
26.0 ± 1.5 35.5 ± 3.7 41.7 ± 4.9 45.6 ± 2.0
0.9 ± 0.9 11.7 ± 1.0 20.9 ± 0.3 26.7 ± 0.2
31.0 ± 1.5 35.2 ± 2.0 44.8 ± 0.6 50.8 ± 1.8
Softwood (China-fir) 30 7.1 60 5.2 90 3.7 120 3.2
15.1 ± 0.3 19.6 ± 0.1 23.4 ± 0.8 24.1 ± 0.9
23.3 ± 1.0 31.9 ± 1.8 38.8 ± 1.3 39.8 ± 1.2
3.3 ± 1.9 6.1 ± 1.4 10.6 ± 2.7 12.6 ± 0.1
21.6 ± 1.6 25.8 ± 2.7 24.9 ± 2.5 31.4 ± 2.7
versed at longer pretreatment times. Although the pretreatment time and substrate species had a little effect on the selective lignin-degrading ability, E. taxodii had selectivity values of 2.0 or higher except for at the end of pretreatment, which indicated that the fungus can degrade lignin selectively without removing large amounts of cellulose. FTIR analysis is a nondestructive technology and requires minimal sample preparation compared to wood component analysis based on the gravimetric techniques, and has been widely used to characterize changes in woods during biological and chemical treatments. Since it is difficult to distinguish clearly biodegradation patterns of pretreated woods from FTIR spectra of woods (the figure not shown), a detailed FTIR analysis was used to describe quantitative changes in lignin and carbohydrate components during the pretreatment based on analysis method of Pandey and Pitman (2003). Table 2 shows the lignin/carbohydrate ratios of the nonpretreated woods were higher than that of pretreated woods in most instances because of the selective removal of lignin, but the extents of decrease in ratios were different for the two woods. This confirmed the above result that E. taxodii had great selective lignindegrading ability. In spite of a little effect of substrate species on the selective ability, E. taxodii produced the selective biodegradation pattern characterized by the preferential removal of lignin during the pretreatment of both hardwood and softwood. Thus, E. taxodii has wide application potential for biological pretreatment of different lignocellulose.
3. Results and discussion 3.1. Evaluation of biodegradation patterns The selective lignin-degrading white-rot fungi could reveal simultaneous degradation of all components on some lignocellulosic substrates, which would affect the efficiency of biological pretreatment. Thus, biodegradation patterns of Chinese willow (hardwood) and China-fir (softwood) by E. taxodii were characterized based on wood component losses and FTIR analysis. As shown in Table 1, E. taxodii had great delignification ability on all woods. The hardwood exhibited higher cellulose losses than the softwood after the 30-days pretreatment, but the cellulose losses of the two woods were always lower than the lignin losses during the pretreatment. The cellulose was barely degraded in comparison with the great removal of lignin (>20%) during the first 30 days. Selective value, the lignin/cellulose loss ratio, was used to evaluate the selective lignin-degrading ability. The selectivity values decreased with increasing pretreatment time. E. taxodii had higher selectivity values on the hardwood than on the softwood at early stages of the pretreatment, but the phenomenon was re-
Table 2 Ratios of the intensity of the lignin associated band to carbohydrate bands. Pretreatment time (day)
Relative intensities of aromatic skeletal vibration (I1506/ against typical bands for carbohydratesa
1510)
I1506/1510/ I1737/1734
I1506/1510/ I1378/1373
I1506/1510/ I1161
I1506/1510/ I898
Hardwood (Chinese willow) 0 0.39 (0.25) 30 0.40 (0.24) 60 0.28 (0.17) 90 0.33 (0.19) 120 0.23 (0.13)
1.00 0.76 0.54 0.63 0.42
(1.10) (0.77) (0.56) (0.67) (0.69)
1.00 0.54 0.40 0.59 0.58
(1.08) (0.59) (0.43) (0.60) (0.63)
1.60 1.68 1.23 1.10 0.77
(2.09) (1.93) (1.48) (1.38) (0.84)
Softwood (China-fir) 0 30 60 90 120
1.80 1.45 1.49 1.35 1.26
(1.83) (1.50) (1.66) (1.45) (1.37)
1.03 0.95 0.92 0.74 0.64
(1.11) (1.03) (1.07) (0.85) (0.79)
4.50 3.87 3.37 3.30 3.14
(3.51) (3.01) (2.97) (3.06) (3.07)
1.82 1.81 1.64 1.69 1.59
(1.11) (1.25) (1.13) (1.14) (1.13)
a Relative intensities were calculated using peak heights (out the parentheses) and areas (in the parentheses).
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3.2. Enzymatic hydrolysis of woods Fig. 1 shows the hydrolysis ratios of woods non-pretreated and pretreated with E. taxodii after 1, 3, 6, 12, 24, 48, 72, 96, 120-h enzymatic hydrolysis. As expected, the non-pretreated hardwood and softwood had very low hydrolysis ratio, but higher hydrolysis ratios were achieved when woods pretreated with E. taxodii were used as the substrate. In most cases, the enzymatic hydrolysis ratios of woods increased with increasing pretreatment time. After the 120-day pretreatment, hydrolysis ratios of hardwood and softwood increased 4.7-fold and 6.3-fold, respectively. Hydrolysis ratios of softwoods were always lower than that of hardwoods. It can be observed that hydrolysis rates of all substrates decreased sharply with increasing hydrolysis time at a given enzyme concentration. Eq. (1) was used to characterize hydrolysis kinetics by regression analysis of the experimental data (dotted curve in Fig. 1). In all cases, a good agreement with the experimental results was obtained (R2 > 0.95) as it is shown by the regression curves in Fig. 1. In this way, the initial hydrolysis rate and the rate of change in the hydrolysis rate can be represented by the kinetic parameters V0 and k. Table 3 shows that biological pretreatment with E. taxodii increased the initial hydrolysis rate V0 of hardwood. The initial hydrolysis rate of the 30-day pretreated hardwood increased 2.1fold, but it had few changes with increasing pretreatment time after 30 days. The initial hydrolysis rate of the pretreated softwood increased only modestly and even decreased after 90-day pretreatment. However, the rate retardation constant k of the two kinds of woods decreased dramatically after the pretreatment, which indicated that biological pretreatment slowed down the decline in hydrolysis rate during enzymatic hydrolysis. After the 120-day pretreatment, k value of hardwood and softwood decreased 6.3fold and 11.7-fold, respectively. Thus, biological pretreatment enhanced the enzymatic hydrolysis of woods mainly by slowing down the decline in hydrolysis rate. 3.3. The effect of biological pretreatment on the initial adsorption of cellulase The adsorption of cellulase to cellulose is the first step of the hydrolysis. The lack of natural substrate accessibility to cellulase may prevent the adsorption of enzyme protein (Desai and Converse, 1997). As shown in Table 4, non-pretreated woods showed very low initial adsorption ratio. But the initial adsorption ratios were increased significantly by biological pretreatment with E. taxodii. After the 90-day pretreatment, the initial adsorption ratios of hardwood and softwood increased 4.6-fold and 5.9-fold,
a
Table 3 Kinetics parameters of enzymatic hydrolysis of hardwood and softwood based on Eq. (1). Pretreatment time (day)
V0 (%/h)
k
R2
Chinese willow (hardwood) 0 30 60 90 120
0.84 2.59 1.72 2.45 2.85
0.73 0.23 0.10 0.13 0.10
0.99 0.99 0.99 0.98 0.98
China-fir (softwood) 0 30 60 90 120
0.50 0.63 0.82 0.44 0.80
2.03 0.68 0.29 0.14 0.16
0.99 0.97 0.98 0.98 0.99
Table 4 Initial adsorption ratio of the hardwood (Chinese willow) and softwood (China-fir) non-pretreated and pretreated with Echinodontium taxodii 2538. Pretreatment time (day)
0 30 60 90
Hardwood (Chinese willow)
Softwood (China-fir)
5.8 ± 1.5 16.3 ± 1.1 22.5 ± 2.2 32.5 ± 5.0
6.2 ± 1.6 24.3 ± 6.4 29.6 ± 2.9 42.8 ± 4.5
respectively. It is well known that woods become more porous during biological pretreatment with white-rot fungi and then cellulase can penetrate further into substrates, so the increase of the initial adsorption ratio may be attributed to the increase of substrate porosity (Wei, 2001; Mooney et al., 1998; Taniguchi et al., 2005). However, increasing the initial adsorption did not always increase the initial hydrolysis rate. For example, compared to the hardwood, the softwood had greater increases in the initial adsorption ratios after the pretreatment, but the initial hydrolysis rate of the softwood increased only modestly. The increase of substrate porosity could enhance the adsorption of cellulase to not only cellulose but also lignin in woods since cellulase can be adsorbed on the lignin by the hydrophobic interaction. But the adsorption of cellulase to the lignin cannot cause the hydrolysis of lignocellulose (Desai and Converse, 1997). Thus, there was no good correlation between the initial hydrolysis rate and the initial adsorption ratio.
b
35
Initial adsorption ratio (%)
18 16
Hydrolysis ratio (%)
Hydrolysis ratio (%)
30 25 20 15 10 5
14 12 10 8 6 4 2
0 0
20
40
60
80
100
Hydrolysis time (hours)
120
0 0
20
40
60
80
100
120
Hydrolysis time (hours)
Fig. 1. Time course of hydrolysis ratio (%) during the hydrolysis of Chinese willow (hardwood, a) and China-fir (softwood, b) non-pretreated (h) and pretreated with Echinodontium taxodii 2538 for 30 (j), 60 (d), 90 (N), 120 (.) days with 20 FPU/g enzyme loading.
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3.4. The effect of biological pretreatment on the adsorption and desorption of cellulase
Table 7 Desorption value and Klason lignin content of the hardwood (Chinese willow) and softwood (China-fir) non-pretreated and pretreated by Echinodontium taxodii 2538.
The adsorption and desorption of cellulase during enzymatic hydrolysis were also investigated to understand further the effect of biological pretreatment on the cellulase–substrate interaction (Gan et al., 2003; Gregg and Saddler, 1996). Generally, enzymatic hydrolysis is considered to consist of the adsorption of cellulase onto the lignocellulose, cellulose hydrolysis and the desorption of cellulase, but only a part of the adsorbed enzyme is desorbed from the residue into the reaction supernatant as the hydrolysis of lignocellulosic substrate proceeds (Gregg and Saddler, 1996). The adsorption of some enzyme proteins was irreversible. As shown in Tables 5 and 6, the added cellulase was adsorbed rapidly onto the substrate during the early stage of hydrolysis. The amount of cellulase adsorbed on the substrate reached the maximum after 12 or 24 h and thenceforth the adsorbed enzymes were released slowly into the supernatant. The maximum of cellulase adsorbed on the pretreated woods was higher than on the non-pretreated woods because of the increase of substrate porosity after the pretreatment. Although most adsorbed proteins still remained associated with substrate residue at the end of enzymatic hydrolysis, it was easier for enzyme adsorbed on pretreated woods to be released into the supernatant. Desorption value was used as an indicator of irreversible adsorption of cellulase in the study (Table 7). The increase of desorption value indicated the decrease of irreversible adsorption. The desorption value of the hardwood and the softwood increased significantly with increasing pretreatment time, which indicated that biological pretreatment reduced the irreversible adsorption of cellulase to wood substrates. After the 90-day pretreatment, the desorption value of the hardwood and the softwood increased 2.0-fold and 9.0-fold, respectively. Interestingly, the rate retardation constant k was very low when the high desorption value was obtained after the pretreatment. The irreversible adsorption of cellulase may lead to enzyme loss in
Pretreatment time (day)
Desorption value
Klason lignin content (%)
Hardwood (Chinese willow) 0 30 60 90
6.3 9.9 18.0 19.1
15.9 ± 0.1 14.0 ± 0.3 13.2 ± 0.8 13.4 ± 1.2
Softwood (China-fir) 0 30 60 90
2.1 14.0 20.1 21.0
32.8 ± 1.0 29.6 ± 0.3 27.8 ± 0.7 26.2 ± 0.4
Table 5 Adsorption ratio of the hardwood (Chinese willow) non-pretreated and pretreated with Echinodontium taxodii 2538 during enzymatic hydrolysis. Hydrolysis time (h)
Adsorption ratio (%) Nonpretreated wood
30-Day pretreated wood
60-Day pretreated wood
90-Day pretreated wood
3 6 12 24 72 120
9.37 ± 1.8 13.0 ± 0.9 39.5 ± 1.9 31.4 ± 3.0 29.3 ± 2.9 33.2 ± 2.7
35.6 ± 3.1 32.4 ± 2.6 38.6 ± 0.9 52.3 ± 4.6 39.4 ± 2.8 42.4 ± 3.6
49.3 ± 1.5 49.6 ± 1.3 62.1 ± 3.2 63.3 ± 5.1 58.8 ± 3.3 45.3 ± 2.1
43.2 ± 1.1 38.1 ± 0.5 54.3 ± 2.2 67.5 ± 0.6 42.4 ± 3.5 48.4 ± 4.7
Table 6 Adsorption ratio of the softwood (China-fir) non-pretreated and pretreated with Echinodontium taxodii 2538 during enzymatic hydrolysis. Hydrolysis time (h)
Adsorption ratio (%) Nonpretreated wood
30-Day pretreated wood
60-Day pretreated wood
90-Day pretreated wood
3 6 12 24 72 120
7.09 ± 1.1 12.6 ± 0.8 25.0 ± 0.2 21.1 ± 1.8 21.6 ± 1.8 22.9 ± 1.5
40.9 ± 1.7 38.9 ± 2.8 52.0 ± 4.3 56.5 ± 2.2 45.9 ± 2.7 42.4 ± 0.7
49.6 ± 2.1 49.2 ± 0.9 61.9 ± 4.7 67.3 ± 5.2 56.2 ± 4.6 47.2 ± 1.3
52.7 ± 1.6 48.0 ± 3.9 65.7 ± 2.3 70.2 ± 1.7 56.5 ± 4.4 49.2 ± 2.8
reaction supernatant and decrease the hydrolysis rate (Gregg and Saddler, 1996). It has been suggested that the improvement of enzymatic hydrolysis was required to achieve effective release and reuse of cellulase (Boussaid and Saddler, 1999). Accordingly, the decrease of irreversible adsorption after biological pretreatment slowed down the decline in hydrolysis rate, leading to the enhancement of enzymatic hydrolysis. Some studies showed the release of adsorbed enzyme could depend partly on the lignin content of the substrate because the binding of cellulase on the lignin was usually irreversible (Gan et al., 2003). As shown in Table 7, the selective lignin-degrading fungus E. taxodii decreased the Klason lignin content of woods after the pretreatment. But a small decrease of the Klason lignin content can result in the very pronounced increase of desorption value, suggesting that irreversible adsorption cannot be explained only by the lignin loss. Lignin structure could also play an important role in the lignin–cellulase interaction. For example, Mooney et al. (1998) reported refiner mechanical pulp (RMP) with sulphonated lignin had higher adsorption capacity than RMP. The binding of cellulase to lignin decreased after the hydrophobic surface of lignin was coated by the surfactant with hydrophilic groups (Eriksson et al., 2002). The structure of lignin macromolecule can be altered by ligninolytic enzymes produced by white-rot fungi during biological pretreatment, such as the change in the content of hydrophilic phenolic hydroxyl groups (Akin et al., 1995; Garzillo et al., 1998; Widsten and Kandelbauer, 2008). Thus, a likely explanation for the decrease of irreversible adsorption is that partial lignin degradation and alteration in lignin structure reduced cellulase adsorption on lignin together after biological pretreatment.
4. Conclusion The research shows that the enzymatic hydrolysis of the hardwood (Chinese willow) and the softwood (China-fir) was enhanced significantly by the biological pretreatment with E. taxodii 2538 which showed great selective lignin-degrading ability on the two woods. Moreover, the pretreatment slowed down the decline in hydrolysis rate during enzymatic hydrolysis by reducing the irreversible adsorption of cellulase. However, there was no good correlation between the initial hydrolysis rate and the initial adsorption ratio. Acknowledgements This work was supported by the Foundation of China’s National Grand Fundamental Research 973 Program (2007CB210200). The authors thank the Centre of Analysis and Test of Huazhong University of Science and Technology for FTIR analysis.
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biortech.2009.05.049. References Akin, D.E., Rigsby, L.L., Sethuraman, A., Morrison, W.H., Gamble, G.R., Eriksson, K.E., 1995. Alterations in structure, chemistry, and biodegradability of grass lignocellulose treated with the white rot fungi Ceriporiopsis subvermispora and Cyathus stercoreus. Appl. Environ. Microbiol. 61, 1591–1598. Amirta, R., Tanabe, T., Watanabe, T., Honda, Y., Kuwahara, M., Watanabe, T., 2006. Methane fermentation of Japanese cedar wood pretreated with a white rot fungus, Ceriporiopsis subvermispora. J. Biotechnol. 123, 71–77. 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. Bioresour. Technol. 58, 241– 245. Blanchette, R.A., 1991. Delignification by wood-decay fungi. Annu. Rev. Phytopathol. 29, 381–403. Boussaid, A., Saddler, J.N., 1999. Adsorption and activity profiles of cellulases during the hydrolysis of two Douglas fir pulps. Enzyme Microb. Technol. 24, 138–143. Desai, S.G., Converse, A.O., 1997. Substrate reactivity as a function of the extent of reaction in the enzymatic hydrolysis of lignocellulose. Biotechnol. Bioeng. 56, 650–655. Eggeman, T., Elander, R.T., 2005. Process and economic analysis of pretreatment technologies. Bioresour. Technol. 96, 2019–2025. Eriksson, T., Börjesson, J., Tjerneld, F., 2002. Mechanism of surfactant effect in enzymatic hydrolysis of lignocellulose. Enzyme Microb. Technol. 31, 353– 364. Gan, Q., Allen, S.J., Taylor, G., 2005. Analysis of process integration and intensification of enzymatic cellulose hydrolysis in a membrane bioreactor. J. Chem. Technol. Biotechnol. 80, 688–698. Gan, Q., Allen, S.J., Taylor, G., 2003. Kinetic dynamics in heterogeneous enzymatic hydrolysis of cellulose: an overview, an experimental. Process Biochem. 38, 1003–1018. Garzillo, A.M.V., Colao, M.C., Caruso, C., Caporale, C., Celletti, D., Buonocore, V., 1998. Laccase from the white-rot fungus Trametes trogii. Appl. Microbiol. Biotechnol. 49, 545–551. 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. Biotechnol. Bioeng. 51, 375–383. Hakala, T.K., Lundell, T., Galkin, S., Maijala, P., Kalkkinen, N., Hatakka, A., 2005. Manganese peroxidases, laccases and oxalic acid from the selective white-rot fungus Physisporinus rivulosus grown on spruce wood chips. Enzyme Microb. Technol. 36, 461–468. Hatakka, A., 2001. Biodegradation of lignin. In: Hofrichter, M., Steinbüchel, A. (Eds.), Lignin, Humic Substances and Coal. Wiley-VCH, Helsinki, pp. 129–167.
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Horwitz, W., 1980. Official Methods of Analysis of the Association of Official Analytical Chemists. Association of Official Analytical Chemists, Washington USA, pp. 134–135. 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. J. Biotechnol. 103, 273–280. 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 Microb. Technol. 25, 644–650. 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. Bioresour. Technol. 64, 113–119. Movagarnejad, K., Sohrabi, M., Kaghazchi, T., Vahabzadeh, F., 2000. A model for the rate of enzymatic hydrolysis of cellulose in heterogeneous solid–liquid systems. Biochem. Eng. J. 4, 197–206. Ohmine, K., Ooshima, H., Harano, Y., 1983. Kinetic study on enzymatic hydrolysis of cellulose by cellulase from Trichoderma viride. Biotechnol. Bioeng. 25, 2041–2053. Pandey, K.K., Pitman, A.J., 2003. FTIR studies of the changes in wood chemistry flowing decay by brown-rot and white-rot fungi. Int. Biodeterior. Biodegrad. 52, 151–160. Pu, Y., Zhang, D., Singh, P.M., Ragauskas, A.J., 2008. The new forestry biofuels sector. Biofuels, Bioprod. Bioref. 2, 58–73. Sun, Y., Cheng, J., 2002. Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresour. Technol. 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. J. Biosci. Bioeng. 100, 637–643. Teymouri, F., Laureano-Perez, L., Alizadeh, H., Dale, B.E., 2005. Optimization of the ammonia fiber explosion (AFEX) treatment parameters for enzymatic hydrolysis of corn stover. Bioresour. Technol. 96, 2014–2018. Tucker, M., Kim, K., Newman, M., Nguyen, Q., 2003. Effects of temperature and moisture on dilute-acid steam explosion pretreatment of corn stover and cellulase enzyme digestibility. Appl. Biochem. Biotechol. 105, 165–177. Van Soest, P.J., 1963. Use of detergents in the analysis of fibrous feeds II: a rapid method for the determination of fibre and lignin. J. Assoc. Off. Agr. Chem. 46, 829–835. Wei, T.C., 2001. The Morphology of Decay by White Rot and Soft Rot Fungi (Bjerkandera adusta, Trametes versicolor, Chaetomium globosum, Daldinia concentrica). College of Environmental Science and Forestry, State University of New York, pp. 19–20. Widsten, P., Kandelbauer, A., 2008. Adhesion improvement of lignocellulosic products by enzymatic pre-treatment. Biotechnol. Adv. 26, 379–386. Yang, B., Wyman, C.E., 2008. Pretreatment: the key to unlocking low-cost cellulosic ethanol. Biofuels, Bioprod. Bioref. 2, 26–40. Zhang, X., Yu, H., Huang, H., Liu, Y., 2007. Evaluation of biological pretreatment with white rot fungi for the enzymatic hydrolysis of bamboo culms. Int. Biodeterior. Biodegrad. 60, 159–164. Zhao, Y., Wang, Y., Zhu, J.Y., Ragauskas, A., Deng, Y., 2008. Enhanced enzymatic hydrolysis of spruce by alkaline pretreatment at low temperature. Biotechnol. Bioeng. 99, 1320–1328.
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The effect of biological pretreatment with the selective white-rot fungus Echinodontium taxodii on enzymatic hydrolysis of softwoods and hardwoods Hongbo Yu a, Guoning Guo b, Xiaoyu Zhang a,*, Keliang Yan a, Chunyan Xu a a b
College of Life Science and Technology, Huazhong University of Science and Technology, Luoyu Road 1037, Wuhan 430074, China Technology Center of Hubei Tobacco (Group) Co. Ltd., Shisheng Road 22, Wuhan 430051, China
a r t i c l e
i n f o
Article history: Received 7 March 2009 Received in revised form 17 May 2009 Accepted 20 May 2009 Available online 9 July 2009 Keywords: Enzymatic hydrolysis Echinodontium taxodii Biological pretreatment Adsorption Wood
a b s t r a c t Selective white-rot fungi have shown potential for lignocellulose pretreatment. In the study, a new fungal isolate, Echinodontium taxodii 2538, was used in biological pretreatment to enhance the enzymatic hydrolysis of two native woods: Chinese willow (hardwood) and China-fir (softwood). E. taxodii preferentially degraded the lignin during the pretreatment, and the pretreated woods showed significant increases in enzymatic hydrolysis ratios (4.7-fold for hardwood and 6.3-fold for softwood). To better understand effects of biological pretreatment on enzymatic hydrolysis, enzyme–substrate interactions were investigated. It was observed that E. taxodii enhanced initial adsorption of cellulase but which did not always translate to high initial hydrolysis rate. However, the rate of change in hydrolysis rate declined dramatically with decreasing irreversible adsorption of cellulase. Thus, the enhancement of enzymatic hydrolysis was attributed to the decline of irreversible adsorption which may result from partial lignin degradation and alteration in lignin structure after biological pretreatment. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Enzymatic hydrolysis is a key step in ethanol production from low-cost lignocellulosic materials (Pu et al., 2008; Zhao et al., 2008). However, the natural resistance of lignocellulose to hydrolysis prevents the release of sugars (Sun and Cheng, 2002; Yang and Wyman, 2008). Some chemical and physicochemical pretreatment processes, such as acid pretreatment, alkaline pretreatment, steam explosion and ammonia fiber explosion, are used to enhance the enzymatic hydrolysis of lignocellulose, but these processes usually require high temperature and operating pressure (Eggeman and Elander, 2005; Teymouri et al., 2005; Tucker et al., 2003). Recently, some studies indicated biological pretreatment could enhance the enzymatic hydrolysis of lignocellulose and had the advantages of mild conditions and low energy consumption (Amirta et al., 2006; Sun and Cheng, 2002). White-rot fungi which can efficiently degrade lignin are usually used in biological pretreatment of lignocellulose (Hakala et al., 2005). Different white-rot fungi vary greatly in the relative rates at which they degrade lignin and carbohydrates in lignocellulose (Hatakka, 2001). The selective lignin-degrading white-rot fungi are considered good candidates for biological pretreatment (Itoh et al., 2003; Taniguchi et al., 2005). In our previous investigation, a new fungal isolate, Echinodontium taxodii 2538, was found to cause selective degradation of lignin * Corresponding author. Tel.: +86 27 87792108; fax: +86 27 87792128. E-mail address: hongbo.fi[email protected] (X. Zhang). 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.05.049
on bamboo culms, and it could significantly enhance the enzymatic hydrolysis of non-woody lignocellulose, such as bamboo culms and corn straw (Zhang et al., 2007). But it is not yet known whether E. taxodii can degrade selectively lignin in woody lignocellulose (wood) and enhance the enzymatic hydrolysis of wood. It is well known that lignocellulose species can also affect the fungal growth and biodegradation patterns (Blanchette, 1991). Accordingly, it is necessary to investigate the biodegradation pattern of wood (including softwood and hardwood) pretreated by E. taxodii and evaluate the effect of the pretreatment on enzymatic hydrolysis in order to fully exploit the potential of the fungus. In the past, some investigators attributed the enhancement of enzymatic hydrolysis after biological pretreatment to the biodegradation of the lignin seal responsible for preventing the penetration of cellulase, but further study is needed as it is likely that fungal delignification also affects the physical properties of the substrate (Taniguchi et al., 2005; Zhang et al., 2007). Enzymatic hydrolysis of insoluble lignocellulose differs considerably from classical solution kinetics (Gan et al., 2005). For instance, in batch reactions enzymatic hydrolysis of lignocellulose has a phase of rapid initial hydrolysis, and this is followed by a sharp decline in hydrolysis rate (Gan et al., 2003; Movagarnejad et al., 2000). Some studies indicated that the low initial adsorption of cellulase to natural substrates could limit the initial hydrolysis rate, and the decline in hydrolysis rate may result from the irreversible adsorption of cellulase during enzymatic hydrolysis (Boussaid and Saddler, 1999; Desai and Converse, 1997). Therefore, fungal
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Nomenclature X V0 t
the hydrolysis ratio (%) the initial hydrolysis rate of enzymatic hydrolysis (%/h) the enzymatic hydrolysis time (h)
pretreatment is likely to affect the cellulase–substrate interaction by altering substrate properties, leading to the enhancement of enzymatic hydrolysis. In this study, E. taxodii was used in biological pretreatment to enhance the enzymatic hydrolysis of common native hardwood and softwood: Chinese willow (Salix babylonica, hardwood) and China-fir (Cunninghamia lanceolata, softwood). The biodegradation patterns of the pretreated woods were also evaluated by wood component losses and Fourier transform infrared (FTIR) analysis. Moreover, adsorption and desorption of cellulase during enzymatic hydrolysis were investigated to better clarify the cause of the improvement of enzymatic hydrolysis.
k
Protein in the centrifugate was measured using the Bradford protein assay (Mooney et al., 1998). Adsorbed cellulase protein was determined based on the difference between the amounts of added protein and protein in the centrifugate. Adsorption ratio was calculated as follows:
Adsorption ratio ð%Þ ¼
Desorption value ¼
Echinodontium taxodii 2538 was found and isolated in Shennongjia Nature Reserve (Hubei, China) (Zhang et al., 2007). Stock culture of the isolate was maintained on potato dextrose agar (PDA) slants at 4 °C. The inoculum was grown on PDA plate at 25 °C for 10 days.
amount of adsorbed protein 100 amount of added protein
In order to evaluate the irreversible adsorption of cellulase, desorption value was used as an indicator in the study. Desorption value was defined as follows:
2. Methods 2.1. Microorganisms and inoculum
the rate retardation constant during enzymatic hydrolysis
maximum of adsorbed protein during hydrolysis amount of adsorbed protein at the end of hydrolysis amount of added protein
The initial adsorption ratio of cellulase to different woods was also determined at a cellulase loading of 20 FPU/g substrate. The initial adsorption reaction was performed in 50 mM sodium acetate buffer (pH 4.8) for 90 min at 4 °C and then for 10 min at 50 °C (Mooney et al., 1999). Protein in the centrifugate was measured after centrifugation at 10,000 rpm for 5 min. Initial adsorption ratio was calculated as follows:
Initial adsorption ratio ð%Þ ¼ 2.2. Biological pretreatment with E. taxodii Chinese willow (Salix babylonica, hardwood) and China-fir (Cunninghamia lanceolata, softwood) woods from Wuhan were ground to pass through a 0.9 mm screen and then were kiln-dried at 60 °C for three days. The biological pretreatments with E. taxodii were carried out in 125-ml Erlenmeyer flasks with 5 g ground woods and 12.5 ml (for hardwood) or 15 ml (for softwood) distilled water. Flasks were sterilized in the autoclave for 20 min at 121 °C and aseptically inoculated with a plug from the plate culture. Cultures were maintained statically at 25 °C for 30, 60, 90 and 120 days, and then dried at 60 °C for three days for enzymatic hydrolysis and chemical analysis. All the cultures were grown in triplicate. A set of non-pretreated sterilized woods were used as control. 2.3. Enzymatic hydrolysis Enzymatic hydrolysis experiments of woods were carried out at 2.5% substrate concentration in 50 mM sodium acetate buffer (pH 4.8) with cellulase (20 FPU/g substrate) at 50 °C. Cellulase was obtained from Sigma. The reaction mixtures were interrupted for different hydrolysis times by the centrifugation at 10,000 rpm for 5 min. The amounts of reducing sugars and protein in the centrifugate were measured. Each time point was analyzed for reducing sugar and protein at least in triplicate.
amount of initial adsorbed protein 100 amount of added protein
2.5. Chemical analysis of woods Dried woods at 60 °C for three days were weighted to determine weight losses. Neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL) and ash in woods were determined according to procedures of Van Soest (1963) and AOAC (Horwitz, 1980). The amount of hemicellulose, cellulose and Klason lignin in woods were calculated as the difference between ADF and NDF, ADF and ADL, ADL and ash, respectively. The selectivity value of lignin degradation was calculated as lignin loss/cellulose loss ratio. The detail process was described as follows: About 1 g of dried wood and 100 ml of neutral detergent solution with sodium lauryl sulphate (SDS) and disodium ethylene diamine tetra acetate (EDTA) were added into a flat bottom flask for the determination of NDF. The mixture was heated to boiling in 5–10 min and refluxed for 60 min from onset of boiling. The hot mixture was filtered through a previously weighed filtering crucible. The residue was rinsed with a minimum of 400 ml hot distilled water and 10 ml acetone, and then was dried at 105 °C until a constant weight was achieved. NDF ð%Þ ¼
Weight of crucible and residue after treating with neutral detergent solution Weight of crucible 100 Weight of wood
2.4. Determination of reducing sugar and cellulase adsorption
ADF was determined using acid detergent solution with cetyl trimethyl ammonium bromide (CTAB) and 0.5 M H2SO4. The procedure was the same as the determination of NDF.
Reducing sugar was measured with the DNS (3,5-dinitrosalicylic acid) reagent (Behera et al., 1996). The hydrolysis ratio was calculated as follows:
ADF ð%Þ ¼
Weight of crucible and residue after treating with neutral detergent solution Weight of crucible 100 Weight of wood
Hydrolysis ratio ð%Þ ¼
amount of reducing sugar produced after enzymatic hydrolysis 0:9 100 amount of cellulose þ amount of hemicellulose
ADF was digested with 72% H2SO4 for 3 h to determine the ADL. After the digestion, the mixture was filtered through the filtering
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crucible and washed with a minimum of 300 ml hot water until acid-free to pH paper. The residue was dried to a constant weight at 105 °C and represents the ADL. ADL ð%Þ ¼
Weight of crucible and residue after treating with 72% H2 SO4 Weight of crucible 100 Weight of wood
The ash was determined as the residue after treatment in the muffle furnace at 575 °C for 24 h.
Ash ð%Þ ¼
Weight of crucible and ash Weight of crucible 100 Weight of wood
Bruker Vertex 70 infrared spectrophotometer was used FTIR analysis of wood samples. The dried woods 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 of Fourier transform infrared spectra were determined by Omnic software. The relative changes in the intensities of lignin peaks at 1506/ 1510 cm1 (hardwood/softwood) against carbohydrate peaks at 1737/1734 (hardwood/softwood), 1378/1373 (hardwood/softwood), 1161 and 898 cm1 were calculated by peak heights and areas (Pandey and Pitman, 2003). The lignin peaks were assigned for aromatic skeletal in lignin. The four carbohydrate peaks were assigned, respectively, for unconjugated C@O in xylans, C–H deformation in cellulose and hemicellulose, C–O–C vibration in cellulose and hemicellulose and C–H deformation in cellulose. 2.6. Data treatment An empirical expression proposed by Ohmine et al. (1983) was used for data treatment of enzymatic hydrolysis, as Eq. (1) shows:
X¼
1 lnð1 þ V 0 k tÞ k
ð1Þ
where X is the hydrolysis ratio (%), V0 is the initial hydrolysis rate (%/h), t is the hydrolysis time (h) and k is the rate retardation constant. According to Eq. (1), initial hydrolysis rate and the rate of change in the hydrolysis rate which was represented by rate retardation constant were determined by regression analysis of the experimental result. Data fit was performed by software Origin 7.5.
Table 1 Percent losses of different components of the hardwood (Chinese willow) and softwood (China-fir) pretreated with Echinodontium taxodii 2538. Pretreatment time (day)
Selectivity value
Component loss (%) Weight
Lignin
Cellulose
Hemicellulose
Hardwood (Chinese willow) 30 28.9 60 3.0 90 2.0 120 1.7
16.0 ± 0.3 22.1 ± 0.1 30.7 ± 0.7 32.5 ± 1.7
26.0 ± 1.5 35.5 ± 3.7 41.7 ± 4.9 45.6 ± 2.0
0.9 ± 0.9 11.7 ± 1.0 20.9 ± 0.3 26.7 ± 0.2
31.0 ± 1.5 35.2 ± 2.0 44.8 ± 0.6 50.8 ± 1.8
Softwood (China-fir) 30 7.1 60 5.2 90 3.7 120 3.2
15.1 ± 0.3 19.6 ± 0.1 23.4 ± 0.8 24.1 ± 0.9
23.3 ± 1.0 31.9 ± 1.8 38.8 ± 1.3 39.8 ± 1.2
3.3 ± 1.9 6.1 ± 1.4 10.6 ± 2.7 12.6 ± 0.1
21.6 ± 1.6 25.8 ± 2.7 24.9 ± 2.5 31.4 ± 2.7
versed at longer pretreatment times. Although the pretreatment time and substrate species had a little effect on the selective lignin-degrading ability, E. taxodii had selectivity values of 2.0 or higher except for at the end of pretreatment, which indicated that the fungus can degrade lignin selectively without removing large amounts of cellulose. FTIR analysis is a nondestructive technology and requires minimal sample preparation compared to wood component analysis based on the gravimetric techniques, and has been widely used to characterize changes in woods during biological and chemical treatments. Since it is difficult to distinguish clearly biodegradation patterns of pretreated woods from FTIR spectra of woods (the figure not shown), a detailed FTIR analysis was used to describe quantitative changes in lignin and carbohydrate components during the pretreatment based on analysis method of Pandey and Pitman (2003). Table 2 shows the lignin/carbohydrate ratios of the nonpretreated woods were higher than that of pretreated woods in most instances because of the selective removal of lignin, but the extents of decrease in ratios were different for the two woods. This confirmed the above result that E. taxodii had great selective lignindegrading ability. In spite of a little effect of substrate species on the selective ability, E. taxodii produced the selective biodegradation pattern characterized by the preferential removal of lignin during the pretreatment of both hardwood and softwood. Thus, E. taxodii has wide application potential for biological pretreatment of different lignocellulose.
3. Results and discussion 3.1. Evaluation of biodegradation patterns The selective lignin-degrading white-rot fungi could reveal simultaneous degradation of all components on some lignocellulosic substrates, which would affect the efficiency of biological pretreatment. Thus, biodegradation patterns of Chinese willow (hardwood) and China-fir (softwood) by E. taxodii were characterized based on wood component losses and FTIR analysis. As shown in Table 1, E. taxodii had great delignification ability on all woods. The hardwood exhibited higher cellulose losses than the softwood after the 30-days pretreatment, but the cellulose losses of the two woods were always lower than the lignin losses during the pretreatment. The cellulose was barely degraded in comparison with the great removal of lignin (>20%) during the first 30 days. Selective value, the lignin/cellulose loss ratio, was used to evaluate the selective lignin-degrading ability. The selectivity values decreased with increasing pretreatment time. E. taxodii had higher selectivity values on the hardwood than on the softwood at early stages of the pretreatment, but the phenomenon was re-
Table 2 Ratios of the intensity of the lignin associated band to carbohydrate bands. Pretreatment time (day)
Relative intensities of aromatic skeletal vibration (I1506/ against typical bands for carbohydratesa
1510)
I1506/1510/ I1737/1734
I1506/1510/ I1378/1373
I1506/1510/ I1161
I1506/1510/ I898
Hardwood (Chinese willow) 0 0.39 (0.25) 30 0.40 (0.24) 60 0.28 (0.17) 90 0.33 (0.19) 120 0.23 (0.13)
1.00 0.76 0.54 0.63 0.42
(1.10) (0.77) (0.56) (0.67) (0.69)
1.00 0.54 0.40 0.59 0.58
(1.08) (0.59) (0.43) (0.60) (0.63)
1.60 1.68 1.23 1.10 0.77
(2.09) (1.93) (1.48) (1.38) (0.84)
Softwood (China-fir) 0 30 60 90 120
1.80 1.45 1.49 1.35 1.26
(1.83) (1.50) (1.66) (1.45) (1.37)
1.03 0.95 0.92 0.74 0.64
(1.11) (1.03) (1.07) (0.85) (0.79)
4.50 3.87 3.37 3.30 3.14
(3.51) (3.01) (2.97) (3.06) (3.07)
1.82 1.81 1.64 1.69 1.59
(1.11) (1.25) (1.13) (1.14) (1.13)
a Relative intensities were calculated using peak heights (out the parentheses) and areas (in the parentheses).
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3.2. Enzymatic hydrolysis of woods Fig. 1 shows the hydrolysis ratios of woods non-pretreated and pretreated with E. taxodii after 1, 3, 6, 12, 24, 48, 72, 96, 120-h enzymatic hydrolysis. As expected, the non-pretreated hardwood and softwood had very low hydrolysis ratio, but higher hydrolysis ratios were achieved when woods pretreated with E. taxodii were used as the substrate. In most cases, the enzymatic hydrolysis ratios of woods increased with increasing pretreatment time. After the 120-day pretreatment, hydrolysis ratios of hardwood and softwood increased 4.7-fold and 6.3-fold, respectively. Hydrolysis ratios of softwoods were always lower than that of hardwoods. It can be observed that hydrolysis rates of all substrates decreased sharply with increasing hydrolysis time at a given enzyme concentration. Eq. (1) was used to characterize hydrolysis kinetics by regression analysis of the experimental data (dotted curve in Fig. 1). In all cases, a good agreement with the experimental results was obtained (R2 > 0.95) as it is shown by the regression curves in Fig. 1. In this way, the initial hydrolysis rate and the rate of change in the hydrolysis rate can be represented by the kinetic parameters V0 and k. Table 3 shows that biological pretreatment with E. taxodii increased the initial hydrolysis rate V0 of hardwood. The initial hydrolysis rate of the 30-day pretreated hardwood increased 2.1fold, but it had few changes with increasing pretreatment time after 30 days. The initial hydrolysis rate of the pretreated softwood increased only modestly and even decreased after 90-day pretreatment. However, the rate retardation constant k of the two kinds of woods decreased dramatically after the pretreatment, which indicated that biological pretreatment slowed down the decline in hydrolysis rate during enzymatic hydrolysis. After the 120-day pretreatment, k value of hardwood and softwood decreased 6.3fold and 11.7-fold, respectively. Thus, biological pretreatment enhanced the enzymatic hydrolysis of woods mainly by slowing down the decline in hydrolysis rate. 3.3. The effect of biological pretreatment on the initial adsorption of cellulase The adsorption of cellulase to cellulose is the first step of the hydrolysis. The lack of natural substrate accessibility to cellulase may prevent the adsorption of enzyme protein (Desai and Converse, 1997). As shown in Table 4, non-pretreated woods showed very low initial adsorption ratio. But the initial adsorption ratios were increased significantly by biological pretreatment with E. taxodii. After the 90-day pretreatment, the initial adsorption ratios of hardwood and softwood increased 4.6-fold and 5.9-fold,
a
Table 3 Kinetics parameters of enzymatic hydrolysis of hardwood and softwood based on Eq. (1). Pretreatment time (day)
V0 (%/h)
k
R2
Chinese willow (hardwood) 0 30 60 90 120
0.84 2.59 1.72 2.45 2.85
0.73 0.23 0.10 0.13 0.10
0.99 0.99 0.99 0.98 0.98
China-fir (softwood) 0 30 60 90 120
0.50 0.63 0.82 0.44 0.80
2.03 0.68 0.29 0.14 0.16
0.99 0.97 0.98 0.98 0.99
Table 4 Initial adsorption ratio of the hardwood (Chinese willow) and softwood (China-fir) non-pretreated and pretreated with Echinodontium taxodii 2538. Pretreatment time (day)
0 30 60 90
Hardwood (Chinese willow)
Softwood (China-fir)
5.8 ± 1.5 16.3 ± 1.1 22.5 ± 2.2 32.5 ± 5.0
6.2 ± 1.6 24.3 ± 6.4 29.6 ± 2.9 42.8 ± 4.5
respectively. It is well known that woods become more porous during biological pretreatment with white-rot fungi and then cellulase can penetrate further into substrates, so the increase of the initial adsorption ratio may be attributed to the increase of substrate porosity (Wei, 2001; Mooney et al., 1998; Taniguchi et al., 2005). However, increasing the initial adsorption did not always increase the initial hydrolysis rate. For example, compared to the hardwood, the softwood had greater increases in the initial adsorption ratios after the pretreatment, but the initial hydrolysis rate of the softwood increased only modestly. The increase of substrate porosity could enhance the adsorption of cellulase to not only cellulose but also lignin in woods since cellulase can be adsorbed on the lignin by the hydrophobic interaction. But the adsorption of cellulase to the lignin cannot cause the hydrolysis of lignocellulose (Desai and Converse, 1997). Thus, there was no good correlation between the initial hydrolysis rate and the initial adsorption ratio.
b
35
Initial adsorption ratio (%)
18 16
Hydrolysis ratio (%)
Hydrolysis ratio (%)
30 25 20 15 10 5
14 12 10 8 6 4 2
0 0
20
40
60
80
100
Hydrolysis time (hours)
120
0 0
20
40
60
80
100
120
Hydrolysis time (hours)
Fig. 1. Time course of hydrolysis ratio (%) during the hydrolysis of Chinese willow (hardwood, a) and China-fir (softwood, b) non-pretreated (h) and pretreated with Echinodontium taxodii 2538 for 30 (j), 60 (d), 90 (N), 120 (.) days with 20 FPU/g enzyme loading.
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3.4. The effect of biological pretreatment on the adsorption and desorption of cellulase
Table 7 Desorption value and Klason lignin content of the hardwood (Chinese willow) and softwood (China-fir) non-pretreated and pretreated by Echinodontium taxodii 2538.
The adsorption and desorption of cellulase during enzymatic hydrolysis were also investigated to understand further the effect of biological pretreatment on the cellulase–substrate interaction (Gan et al., 2003; Gregg and Saddler, 1996). Generally, enzymatic hydrolysis is considered to consist of the adsorption of cellulase onto the lignocellulose, cellulose hydrolysis and the desorption of cellulase, but only a part of the adsorbed enzyme is desorbed from the residue into the reaction supernatant as the hydrolysis of lignocellulosic substrate proceeds (Gregg and Saddler, 1996). The adsorption of some enzyme proteins was irreversible. As shown in Tables 5 and 6, the added cellulase was adsorbed rapidly onto the substrate during the early stage of hydrolysis. The amount of cellulase adsorbed on the substrate reached the maximum after 12 or 24 h and thenceforth the adsorbed enzymes were released slowly into the supernatant. The maximum of cellulase adsorbed on the pretreated woods was higher than on the non-pretreated woods because of the increase of substrate porosity after the pretreatment. Although most adsorbed proteins still remained associated with substrate residue at the end of enzymatic hydrolysis, it was easier for enzyme adsorbed on pretreated woods to be released into the supernatant. Desorption value was used as an indicator of irreversible adsorption of cellulase in the study (Table 7). The increase of desorption value indicated the decrease of irreversible adsorption. The desorption value of the hardwood and the softwood increased significantly with increasing pretreatment time, which indicated that biological pretreatment reduced the irreversible adsorption of cellulase to wood substrates. After the 90-day pretreatment, the desorption value of the hardwood and the softwood increased 2.0-fold and 9.0-fold, respectively. Interestingly, the rate retardation constant k was very low when the high desorption value was obtained after the pretreatment. The irreversible adsorption of cellulase may lead to enzyme loss in
Pretreatment time (day)
Desorption value
Klason lignin content (%)
Hardwood (Chinese willow) 0 30 60 90
6.3 9.9 18.0 19.1
15.9 ± 0.1 14.0 ± 0.3 13.2 ± 0.8 13.4 ± 1.2
Softwood (China-fir) 0 30 60 90
2.1 14.0 20.1 21.0
32.8 ± 1.0 29.6 ± 0.3 27.8 ± 0.7 26.2 ± 0.4
Table 5 Adsorption ratio of the hardwood (Chinese willow) non-pretreated and pretreated with Echinodontium taxodii 2538 during enzymatic hydrolysis. Hydrolysis time (h)
Adsorption ratio (%) Nonpretreated wood
30-Day pretreated wood
60-Day pretreated wood
90-Day pretreated wood
3 6 12 24 72 120
9.37 ± 1.8 13.0 ± 0.9 39.5 ± 1.9 31.4 ± 3.0 29.3 ± 2.9 33.2 ± 2.7
35.6 ± 3.1 32.4 ± 2.6 38.6 ± 0.9 52.3 ± 4.6 39.4 ± 2.8 42.4 ± 3.6
49.3 ± 1.5 49.6 ± 1.3 62.1 ± 3.2 63.3 ± 5.1 58.8 ± 3.3 45.3 ± 2.1
43.2 ± 1.1 38.1 ± 0.5 54.3 ± 2.2 67.5 ± 0.6 42.4 ± 3.5 48.4 ± 4.7
Table 6 Adsorption ratio of the softwood (China-fir) non-pretreated and pretreated with Echinodontium taxodii 2538 during enzymatic hydrolysis. Hydrolysis time (h)
Adsorption ratio (%) Nonpretreated wood
30-Day pretreated wood
60-Day pretreated wood
90-Day pretreated wood
3 6 12 24 72 120
7.09 ± 1.1 12.6 ± 0.8 25.0 ± 0.2 21.1 ± 1.8 21.6 ± 1.8 22.9 ± 1.5
40.9 ± 1.7 38.9 ± 2.8 52.0 ± 4.3 56.5 ± 2.2 45.9 ± 2.7 42.4 ± 0.7
49.6 ± 2.1 49.2 ± 0.9 61.9 ± 4.7 67.3 ± 5.2 56.2 ± 4.6 47.2 ± 1.3
52.7 ± 1.6 48.0 ± 3.9 65.7 ± 2.3 70.2 ± 1.7 56.5 ± 4.4 49.2 ± 2.8
reaction supernatant and decrease the hydrolysis rate (Gregg and Saddler, 1996). It has been suggested that the improvement of enzymatic hydrolysis was required to achieve effective release and reuse of cellulase (Boussaid and Saddler, 1999). Accordingly, the decrease of irreversible adsorption after biological pretreatment slowed down the decline in hydrolysis rate, leading to the enhancement of enzymatic hydrolysis. Some studies showed the release of adsorbed enzyme could depend partly on the lignin content of the substrate because the binding of cellulase on the lignin was usually irreversible (Gan et al., 2003). As shown in Table 7, the selective lignin-degrading fungus E. taxodii decreased the Klason lignin content of woods after the pretreatment. But a small decrease of the Klason lignin content can result in the very pronounced increase of desorption value, suggesting that irreversible adsorption cannot be explained only by the lignin loss. Lignin structure could also play an important role in the lignin–cellulase interaction. For example, Mooney et al. (1998) reported refiner mechanical pulp (RMP) with sulphonated lignin had higher adsorption capacity than RMP. The binding of cellulase to lignin decreased after the hydrophobic surface of lignin was coated by the surfactant with hydrophilic groups (Eriksson et al., 2002). The structure of lignin macromolecule can be altered by ligninolytic enzymes produced by white-rot fungi during biological pretreatment, such as the change in the content of hydrophilic phenolic hydroxyl groups (Akin et al., 1995; Garzillo et al., 1998; Widsten and Kandelbauer, 2008). Thus, a likely explanation for the decrease of irreversible adsorption is that partial lignin degradation and alteration in lignin structure reduced cellulase adsorption on lignin together after biological pretreatment.
4. Conclusion The research shows that the enzymatic hydrolysis of the hardwood (Chinese willow) and the softwood (China-fir) was enhanced significantly by the biological pretreatment with E. taxodii 2538 which showed great selective lignin-degrading ability on the two woods. Moreover, the pretreatment slowed down the decline in hydrolysis rate during enzymatic hydrolysis by reducing the irreversible adsorption of cellulase. However, there was no good correlation between the initial hydrolysis rate and the initial adsorption ratio. Acknowledgements This work was supported by the Foundation of China’s National Grand Fundamental Research 973 Program (2007CB210200). The authors thank the Centre of Analysis and Test of Huazhong University of Science and Technology for FTIR analysis.
H. Yu et al. / Bioresource Technology 100 (2009) 5170–5175
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