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Combination of liquid hot water pretreatment and wet disk milling to improve the efficiency of the enzymatic hydrolysis of eucalyptus
Bioresource Technology 128 (2013) 725–730
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Combination of liquid hot water pretreatment and wet disk milling to improve the efficiency of the enzymatic hydrolysis of eucalyptus Wei Weiqi a, Wu Shubin a,⇑, Liu Liguo b a b
State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, China Guangzhou You Rui Bio-Technology Limited Company, Guangzhou 510640, China
h i g h l i g h t s " Two environmentally friendly pretreatments were combined to enhance the sugar yield. " The concentration of fermentation inhibitors in the prehydrolyzate are discussed. " Compositions of water-insoluble solids resulting from pretreatment are discussed. " Find out the optimal pretreatment conditions basis on the total sugars yield. " A further analysis of insoluble solid resulting from enzymatic hydrolysis.
a r t i c l e
i n f o
Article history: Received 7 May 2012 Received in revised form 26 August 2012 Accepted 27 August 2012 Available online 7 September 2012 Keywords: Eucalyptus Liquid hot water pretreatment Wet disk milling Enzymatic hydrolysis Sugar recovery yield
a b s t r a c t Combination of liquid hot water pretreatment (LHWP) and wet disk milling (WDM) was investigated in this study to enhance the sugar recovery yield both in prehydrolyzate and enzymatic hydrolyzate. The results show that WDM with LHWP at 180 °C for 20 min produced maximum xylose and glucose yields of 91.62% and 88.12%, respectively, which are higher than that of dilute acid pretreatment or individual LHWP. Corresponding concentration of fermentation inhibitors such as acetic acid, HMF, and furfural in the prehydrolyzate are about 0.98, 0.07 and 0.78 g/L, respectively, which indicated that the detoxification may be not required in the next fermentation step. The acid-insoluble lignin recovery in the insoluble solid resulting from enzymatic hydrolysis, was 25.67/100 g raw material, representing 90.7% of acidinsoluble lignin in the eucalyptus biomass. It can be concluded that liquid hot water pretreatment combined with wet disk milling can be successfully applied to eucalyptus. Ó 2012 Published by Elsevier Ltd.
1. Introduction Due to a lack of resource sustainability of fossil fuels as well as negative environmental effects from emissions, research is being done to find a liquid fuel for use as a gasoline replacement (Arthur et al., 2011). Lignocellulosic materials are the most abundant biomass available to the world (Buranov and Mazza, 2008). The polysaccharides present in the lignocellulosic materials can be depolymerized to monosaccharide and then converted to ethanol via appropriate processes (Shuai et al., 2010). However, the natural structures of lignocellulosic materials make it hard for microorganism to utilize this material to produce ethanol. Therefore, efficient pretreatment method is needed so that sequential enzymatic hydrolysis gives maximal sugar productivity and at the same time any loss of sugar is minimized (Jorgensen et al., 2007).
⇑ Corresponding author. E-mail address: [email protected] (W. Shubin). 0960-8524/$ - see front matter Ó 2012 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.biortech.2012.08.130
Pretreatment techniques to overcome this recalcitrance are essential for saccharification and bioethanol production from lignocelluloses (Hendriks and Zeeman, 2008) in future biorefineries (Himmel et al., 2007). Various pretreatments using sulfuric acid, hydrochloric acid, formic acid and alkali reagents have been attempted (Cara et al., 2008; Vanderghem et al., 2012; Zhu and Pan, 2010). However, pretreatments using these chemicals have some problems such as the formation of inhibiting compounds for saccharification and subsequent ethanol fermentation and the pH requirements for downstream processes (Sun and Cheng, 2002). Moreover, the use of strong acids such as sulfuric acid in pretreatments has significant environmental risks. The development of environmentally friendly pretreatments that do not involve chemicals, such as ball milling (Endo et al., 2006), wet disk milling (Hideno et al., 2012) and liquid hot water pretreatment (Goh et al., 2010; Liu and Wyman, 2005), has been studied. However, they also have disadvantages. For example, ball milling has a high energy requirement and is not economically feasible in general (Inoue et al., 2008). Liquid hot water pretreatment,
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in which pressure is utilized to maintain water in the liquid state at elevated temperatures, has been reported to have the potential to remove most hemicelluloses, but the water-insoluble solid resulting from pretreatment are not very good for enzyme hydrolysis (Yu et al., 2010). To eliminate these disadvantages, Inoue et al. investigated ball milling combined with hot-compressed water treatment of eucalyptus, which can save energy for pretreatment and enzyme loading for enzymatic hydrolysis (Inoue et al., 2008). However, there have been very few reports about wet disk milling combined with liquid hot water pretreatment (LHWP), especially, for eucalyptus raw material. The objective of this work is to enhance the sugar recovery yield both in prehydrolyzate and enzymatic hydrolyzate through combination of liquid hot water pretreatment (LHWP) and wet disk milling (WDM). In addition, the compositions of water-insoluble solids resulting from liquid hot water pretreatment and the concentration of fermentation inhibitors such as acetic acid, HMF, and furfural in the prehydrolyzate are also discussed. Finally, there is a further analysis of insoluble solid resulting from enzymatic hydrolysis, found that most of the acid insoluble lignin can be recycled in the enzymatic hydrolysis residues. 2. Methods 2.1. Raw material The eucalyptus chips used in this study was provided by a local factory near LongYan, FuJian, China. The raw materials were airdried and then milled to give a size less than 5 mm by a Rub silk machine. The chemical composition of the raw material (on a dry weight basis) was 42.6% glucose, 15.4% xylose, 28.3% acid-insoluble lignin, 2.4% acid-soluble lignin, 3.3% extractives and 8.0% others. 2.2. Liquid hot water pretreatment Liquid hot water pretreatment was performed in a laboratory scale stirred autoclave. The reactor has a total volume of 1 L, with an electric heater and magnetic agitation. Temperature and agitation speed during the pretreatment was controlled and monitored with a modular controller (Perez et al., 2008). The amount of dry feedstock loaded was 75 g and water was added at 1/10 (w/v) solid/liquid ratio. Both water and raw material were initially at room temperature. Agitation was set at 500 rpm. The other conditions of pretreatment are summarized in Table 1. The pretreatment temperatures were designed within the range of 160–200 °C, with a residence time from 0 to 40 min. When the pretreatment was finished, the reactor was immediately cooled down by cooling water and then removed from heating jacket. The pretreated solution was then separated by filtration. The liquid fraction was analyzed by Table 1 Water-insoluble solids (WIS) fraction composition and recovery yield after liquid hot water pretreatment of eucalyptus under different pretreatment conditions. Conditions
Gravimetric recovery (%)
WIS composition (%)
20
87.28 82.48 77.66 74.24 71.35
44.83 46.91 51.38 53.41 53.96
0 10 20 30 40
80.87 78.29 77.66 76.89 75.39
45.35 49.47 52.98 53.61 54.48
Temp (°C)
Time (min)
160 170 180 190 200 180
Cellulose
HCel
AIL
ASL
Other
9.76 6.86 2.44 1.38 0.85
31.71 32.28 33.25 32.92 33.60
2.75 2.42 2.06 1.59 1.08
10.95 11.53 10.85 10.70 10.51
10.53 6.65 2.44 2.41 1.54
31.91 32.09 33.25 33.54 34.11
2.75 2.49 2.06 1.97 1.89
9.46 9.30 9.25 8.37 7.81
high-pressure liquid chromatography (HPLC) and ion chromatography (IC) to determine the concentration of inhibitors and sugars in the prehydrolyzate. The yield of water-insoluble solids (WIS) fraction was measured and then was stored in freezer for next step. 2.3. Wet disk milling pretreatment Wet disk milling was performed by a method based on previous reports (Endo et al., 2008; Hideno et al., 2009), using the Supermasscolloider MKZA10 (Masuko Sangyo Co., Ltd., Saitama, Japan) equipped with two ceramic nonporous disk grinders, which are adjustable for a clearance of 20–40 lm between the upper and lower grinders. The two disk grinders revolved at 1800 rpm. The liquid hot water pretreated residues were diluted with deionized water to 1/10 (w/v) solid/liquid ratio and then thrown into the machine, the operation was repeated 15 times. Slurry samples were taken after each operation and the moisture content of the samples was analyzed. 2.4. Enzymatic hydrolysis The enzymatic hydrolysis was then performed in 250 mL flasks using 50 mM sodium acetate buffer (pH 4.8) and 2% dry matter (w/ w) at 50 °C on an orbital shaker at 150 rpm for 60 h. The pretreated solid used for enzymatic hydrolysis was not extra handled by washing and drying process in this study. For preparing a 2% dry mass slurry, It first measured the moisture content of pretreated solid and then weighed the pretreated solid in wet basis which contained 2 g of dry mass. The pretreated solid weighed was further mixed with sodium acetate buffer with a final volume of 100 mL for enzymatic hydrolysis. The Cellulose enzyme was provided by Genencor, with a filter paper activity of 60 FPU/mL. The cellulase loading was set at 20 FPU/g dry matter. After the enzymatic hydrolysis test was completed, the sugars in enzyme hydrolyzate were analyzed by ion chromatography. 2.5. Analytical methods The chemical composition of raw material and water-insoluble solids (WIS) fraction resulting from liquid hot water pretreatment was determined by a standard analysis procedure of biomass composition, which was modified by the National Renewable Energy Laboratory (NREL) analytic methods (Cheng et al., 2006). All liquid samples from the pretreatment and enzymatic hydrolysis tests were filtered using a 0.45 lm filter and then diluted appropriately with the deionized water. The concentration of xylose, xylo-oligosaccharides, glucose and gluco-oligosaccharides were quantitatively analyzed by ion chromatography system (CarboPACTM PA1 column, ED detector, column temperature 30 °C, velocity 0.65 ml/min). The oligosaccharides in the liquid fraction were back-calculated after a secondary hydrolysis into monomer sugars with 4% sulfuric acid. The concentration of acetic acid, furfural and HMF were quantitatively analyzed at 45 °C using a HPLC system (Agilent 1200 series, Agilent Technologies) equipped with a refractive index detector. The sample separation was performed using a polystyrene–sulfonic acid column (Coregel-87H3 column, Transgenomic Technologies) at 65 °C with 4 mM H2SO4 as the eluent at a flow rate of 1.0 mL min 1 (Hsu et al., 2010). 3. Results and discussion 3.1. Compositions of WIS resulting from LHWP The total gravimetric recovery (solids remaining after liquid hot water pretreatment divided by original oven-dried weight) and the
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composition of water-insoluble solids resulting from liquid hot water pretreatment are summarized in Table 1. From the table, it can know that cellulose and acid-insoluble lignin is the major chemical composition of WIS, the content of hemicelluloses, ASL, and other material is relatively small. Part of the material was dissolved during liquid hot water pretreatment. As expected, with the rise of pretreatment temperature, the total gravimetric recovery is decreased. For example, when the pretreatment temperature is raised from 160 to 200 °C, corresponding gravimetric recoveries is decreased from 87.28% to 71.35%. In contrast, pretreatment time seems to exert a limited influence on total gravimetric recovery at a constant temperature. With the increase of pretreatment temperature or time, the cellulose content of WIS was increased. For example, when the pretreatment temperature is raised from 160 to 200 °C, corresponding cellulose content is increased from 44.83% to 53.96%. The cellulose content of WIS was always higher than the initial percentage of 42.6%, this is because the solubilization of water-soluble biomass components into prehydrolyzate resulted in a cellulose enrichment in WIS fraction. The acid-insoluble lignin content of WIS increased as pretreatment temperature or time increased, representing between 31% and 35% of the pretreated material. Referred to the initial content in raw material by considering the total gravimetric recovery, were 24–27%, which means that the pretreatment results in an acid-insoluble lignin degradation when compared to the original material (initial acid-insoluble lignin content in raw material 28.3%). Similar trends are reported in a study on the liquid hot water pretreatment with flowing hot water through cellulosic biomass in a small tubular flow through reactor by Liu and Wyman (2003, 2004a, 2004b). Moreover, interestingly, acid soluble lignin (ASL) demonstrated a decreased trend with the increase of pretreatment temperature or time, it is likely that the hot water solubilized part of the ASL, since hot water liberates acids and facilitates the breakage of such ether linkages in biomass during biomass hydrolysis (Wang et al., 2012). In addition, with the increase of pretreatment temperature or time, significant amounts of hemicellulose were degraded during liquid hot water pretreatment. For example, when the pretreatment temperature is above 200 °C, corresponding hemicellulose degradation is more than 97%, which implies that there was a nearly complete hydrolysis of hemicellulose during pretreatment under high severity conditions. The release of hemicallulose from the raw material during pretreatment can improve the efficiency of subsequent enzymatic hydrolysis (Zhu et al., 2012). 3.2. Prehydrolyzate composition Table 2 shows the composition of the prehydrolyzate obtained after liquid hot water pretreatment based on 100 g raw material. From the table, it can know that xylose and xylo-oligosaccharides
is the major carbohydrate in the prehydrolyzate liquor. The highest yield of total xylose in the prehydolyzate was 13.09 g (combined xylose and xylo-oligosaccharides, 3.39 and 9.70 g, respectively), representing 85% of total xylose in the eucalyptus biomass, which was generated after a pretreatment at 180 °C and 20 min residence time. Moreover, limited amounts of glucose and gluco-oligosaccharides were also released from cellulose during the liquid hot water pretreatment. The amount of total glucose (combined glucose and gluco-oligosaccharides) release was generally below 2.5 g per 100 g raw material. These results support the observation reported by others that the decomposition of hemicellulose starts at a lower temperature (180 °C) than that of cellulose in the liquid hot water. In addition, some furan byproducts from the conversion of sugars, such as furfural and HMF (hydroxymethylfurfural), were observed in the prehydrolyzate after pretreatment. Acetic acid was also detected in prehydrolyzate and believed to be generated from the release of the acetyl groups present in the hemicelluloses. These byproducts as mentioned above may have an inhibitory effect on the growth of xylose-fermenting microorganisms and reduce ethanol production. Fig. 1 shows the yield of sugars in the prehydrolyzate fractions as a function of pretreatment temperature or time. As expected, with the increase of pretreatment temperature or time, the yield of total xylose in prehydrolyzate is increase first, and then decrease. For example, when the pretreatment temperature is raised from 160 to 180 °C, corresponding yield of total xylose in prehydrolyzate is increased from 30.8% to 85.0%, continues to increase temperature, the yield of total xylose is quickly decreased. This decrease of total xylose in the prehydrolyzate can most likely be attributed to the degradation of xylose into furfural as a result of severe pretreatment conditions. The yield of xylo-oligosaccharides increased significantly during the first 20 min, at 180 °C, and then decreased quickly after about 20 min, which suggested that more xylo-oligosaccharides is hydrolyzed into xylose monomer for a long residence time or under more severe conditions. By contrast, the recovery yield of xylose monomer was increased continually from 4.03% to 26.36% with pretreatment residence time increased from 0 to 40 min, which suggested that the rate of xylo-oligosaccharides hydrolyzed into xylose monomer is much faster than that of xylose monomer hydrolyzed into furfural. Fig. 1 also shows that more xylose is oligomeric at a relatively low temperature. For example, the yield of xylo-oligosaccharides at 160 °C was 3.66 g, representing 77.22% of total xylose in the prehydrolyzate, and the yield of xylo-oligosaccharides at 200 °C was 4.23 g, representing 50.59% of total xylose in the prehydrolyzate. The yield of glucose and gluco-oligosaccharides in the liquid fractions was increased only slightly, which indicated that eucalyptus cellulose is recalcitrant to solubilization at these temperatures. Fig. 2 shows the yield of the fermentative inhibitors such as acetic acid, furfural and HMF at various pretreatment conditions. From
Table 2 Composition of the prehydrolyzate after liquid hot water pretreatment of eucalyptus under different pretreatment conditions. Conditions
Sugar analysis (g per 100 g raw material)
Inhibitor analysis (g/L)
Temp (°C)
Time (min)
Xylose monomer
Xylooligosaccharides
Total xylose
Glucose monomer
Glucooligosaccharides
Total glucose
Acetic acid
HMF
Furfural
160 170 180 190 200
20
1.08 2.02 3.39 4.03 4.13
3.66 6.36 9.70 5.14 4.23
4.74 8.38 13.09 9.17 8.36
nd 0.12 0.07 0.35 0.68
nd 0.36 0.63 1.00 1.65
nd 0.48 0.70 1.35 2.33
0.34 0.78 0.98 1.32 2.21
nd nd 0.07 0.17 044
0.24 0.44 0.78 2.61 3.29
180
0 10 20 30 40
0.62 1.23 3.39 3.45 4.06
4.26 9.85 9.70 7.92 5.51
4.88 11.08 13.09 11.37 9.57
nd nd 0.07 0.13 0.15
0.18 0.30 0.63 0.71 0.92
0.18 0.30 0.70 0.84 1.07
0.38 0.64 0.98 1.18 1.84
nd nd 0.07 0.09 0.08
0.12 0.42 0.78 1.66 2.05
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Yield of xylose / (%)
60
30 20
40
10
20 0
4
Residence time:20min 40
A
Yield of glucose / (%)
Xylo-oligosaccharides Xylose-monomer Total xylose Gluco-oligosaccharides Glucose-moomer Total glucose
80
0
160 170 180 190 Temperature of LHW pretreatment /
The concentration of inhibitors (g/L)
728
200
A Residence time:20min
3
Furfural 5-HMF Acetic acid
2
1
0 160
Temperature:180
60
30
40
20 Gluco-oligosaccharides Glucose-monomer Total glucose
20 0
10
B 10 30 0 20 Residence time of LHW pretreatment /
200
40
0 40
Fig. 1. Yields of sugars in the prehydrolyzate fraction resulting from different pretreatment conditions. (A) Pretreatment temperature and (B) pretreatment time.
Fig. 2, it can know that furfural which is generated by conversion from xylose is increased as the rise of both pretreatment temperature and time. For example, when the pretreatment temperature is raised from 160 to 200 °C, corresponding furfural content is increased from 0.24 to 3.29 g/L, which indicates that more xylose decomposition took place under severe conditions. Another important inhibitor, acetic acid, which is generated by the hydrolysis of the acetyl groups on hemicellulose is also increased with the strengthening of both pretreatment temperature and time. For example, when the pretreatment time is raised from 0 to 40 min, corresponding acetic acid content in the prehydrolyzate is increased from 0.38 to 1.84 g/L. In this study, when the yield of total xylose in the prehydolyzate was about 85%, corresponding concentrations of acetic acid, HMF, and furfural in the prehydrolyzate are about 0.98, 0.07 and 0.78 g/L, respectively. Previous studies have indicated that a decline in the ethanol yield from xylose fermentations when the concentration of acetic acid, HMF, and furfural was above 2.0, 1.0, and 1.0 g/L, respectively (Palmqvist and HahnHägerdal, 2000). Therefore, the detoxification methods, such as over-liming, vacuum evaporation and activated charcoal, may be not required to diminish the inhibition effect in next step of fermentation. 3.3. Enzymatic hydrolysis Table 3 shows the composition of the hydrolyzate obtained after enzymatic hydrolysis based on 100 g raw material. From the table it can know that glucose is the major carbohydrate in the enzyme hydrolyzate. With the increase of pretreatment temperature, there is a discernible upward trend in the yield of glucose. For example, when the pretreatment temperature is raised from 170 to 200 °C, corresponding glucose yield in enzymatic
The concentration of inhibitors (g/L)
Yield of xylose / (%)
80
Yield of glucose / (%)
Xylo-oligosaccharides Xylose-monomer Total xylose
190 180 170 Temperature of LHW pretreatment /
3
B Temperature:180 Furfural Acetic acid 5-HMF
2
1
0 0
30 20 10 Residence time of LHW pretreatment /
40
Fig. 2. Concentration of fermentation inhibitors in the prehydrolyzate resulting from different pretreatment conditions. (A) Pretreatment temperature and (B) pretreatment time.
Table 3 The yield of xylose, glucose and acid-insoluble lignin both in enzyme hydrolyzate and enzyme scraps under different pretreatment conditions (g per 100 g raw material). Optimal conditions
Enzyme hydrolyzate
Enzymatic hydrolysis residue
Xylose
Glucose
AIL
Xylose
Glucose
170 °C, 20 min 180 °C, 20 min 190 °C, 20 min 200 °C, 20 min 180 °C, 20 min + Wet disk milling
1.68 0.84 0.65 0.32 1.02
24.67 29.83 32.17 34.12 36.84
26.63 25.82 24.44 23.98 25.67
3.98 0.43 0.08 nd nd
14.02 10.07 7.48 4.38 3.09
hydrolyzate is increased from 24.67 to 34.12 g. Similar trends are reported in previous literature (Zhu et al., 2012). This upward trend can be explained by the fact that hemicellulose removal during the pretreatment in the form of either xylose or furfural can lead to high glucose yield in the hydrolyzate after the enzymatic hydrolysis. However, it was regretful to note that when the highest yield of total xylose in the prehydolyzate was about 85%, corresponding glucose yield in enzymatic hydrolyzate is only about 29.83 g (representing 70.02% of glucose in the eucalyptus biomass). Therefore, the post-pretreatment such as wet disk milling, are used to improve the reactivity of the water-insoluble solids with respect to enzymatic hydrolysis in this study. Related results are also summarized in Table 3, it was easy to find that the glucose yield in enzymatic hydrolyzate increased significantly when liquid hot water
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W. Weiqi et al. / Bioresource Technology 128 (2013) 725–730 Table 4 Yields of glucose and xylose from both liquid hot water pretreatment and enzymatic hydrolysis under optimal conditions (g per 100 g raw material).
c
Stage1a
Stage2b
Stage(1 + 2)c
Xylose
Glucose
Xylose
Glucose
Xylose
Glucose
170 °C, 20 min 180 °C, 20 min 190 °C, 20 min 200 °C, 20 min 180 °C, 20 min + Wet disk milling
8.38 13.09 9.17 8.36 13.09
0.48 0.70 1.35 2.33 0.70
1.68 0.84 0.65 0.32 1.02
24.67 29.83 32.17 34.12 36.84
10.06 13.93 9.82 8.68 14.11
25.15 31.18 33.52 36.45 37.54
Glucose and xylose in the prehydrolyzate. Glucose and xylose in the enzyme hydrolyzate. Glucose and xylose both in prehydrolyzate and enzyme hydrolyzate.
Xylose in prehydrolyzate The recovery yield of xylose / (%)
a b
Optimal conditions
100
Xylose in enzyme hydrolyzate
80
60
40
20
3.4. Total sugars Overall sugar yield, calculated in relation to the raw material for the two phases: pretreatment and enzymatic hydrolysis, is a major indicator of the potential amount of sugar that could be used for ethanol production. Therefore, in this study, the total sugars available for fermentation from eucalyptus are the summation of the xylose and glucose found in both the prehydrolyzate and the enzymatic hydrolyzate. The yields of glucose and xylose from both pretreatment and enzymatic hydrolysis are summarized in Table 4 and Fig. 3. From the table and figure, it can know that on the basis of 100 g raw material, the maximum xylose yield (combined xylose monomer and xylo-oligosaccharides) was about 14.11 g, representing 91.6% of total xylose in the eucalyptus biomass. This result compares favorably with that obtained when the same raw material was pretreated just with dilute sulfuric acid, where a maximum total xylose yield of 80.1% was attained (Wei et al., 2012). This improvement of xylose yield was attributed to the less degradation of xylose into furfural in the prehydrolyzate. On the basis of
18 W 0 et , di 20m sk i m n+ ill in g
20 0
,2 0m
in
,2 0m in 19 0
17 0
18 0
,2 0m
,2 0m
in
in
0
100
Glucose in prehydrolyzate The recovery yield of glucose / (%)
Glucose in enzyme hydrolyzate
80
60
40
20
20 0
18 W 0 et , di 20m sk i m n+ i ll in g
in ,2 0m
in 0m ,2 19 0
18 0
,2 0m
,2 0m
in
in
0
17 0
pretreatment (LHWP) and wet disk milling (WDM) were combined compared with the individual liquid hot water pretreatment. For example, the glucose yield from the wet disk milled sample followed LHWP were 36.84 g (representing 86.48% of glucose in the eucalyptus biomass), which is obvious higher than that from the individual LHWP sample (29.83 g, representing 70.02% of glucose in the eucalyptus biomass). This increase of glucose yield after wet disk milling can most likely be attributed to the fiberization of cellulose by WDM. After the enzymatic hydrolysis, the insoluble solid resulting from enzymatic hydrolysis were treated with two step acid hydrolysis to test the remaining sugars and acid insoluble lignin (AIL) content. Related data were summarized in Table 3. It was easy to find an inverse proportion between the glucose yields from the enzymatic hydrolysis and the AIL content of the pretreated solid residue. For example, when the AIL content in pretreated solid residue is decreased from 26.63 to 23.98 g, corresponding yield of glucose in enzymatic hydrolyzate is increased from 24.67 to 34.12 g. This result implies that the lignin content of the pretreated solid residue is a critical factor to affect the performance of the enzymatic hydrolysis reaction in this study. Similar trends are reported in previous literature (Wei et al., 2012). Contrast the AIL yield in enzymatic hydrolysis residue, we can know that the lignin has not been degraded during the wet disk milling pretreatment. This is because the pretreatment of WDM is only a physical process, neither acids nor alkalis have been used in this process. Moreover, from Table 3, it can also know that, under the optimal pretreatment condition, the acid-insoluble lignin recovery in the enzymatic hydrolysis residue was about 25.67 g, representing 90% of acidinsoluble lignin in the eucalyptus biomass.
Fig. 3. The recovery yields of glucose and xylose from both pretreatment and enzymatic hydrolysis.
100 g raw material, the maximum glucose yield was about 37.54 g, representing 88.12% of total glucose in the eucalyptus biomass. This result compared with the individual liquid hot water pretreatment, where the highest glucose recovery yield was only about 73.19%, indicated that wet disk milling can greatly improve the reactivity of the water-insoluble solids with respect to enzymatic hydrolysis in this study.
4. Conclusions Wet disk milling (WDM) with liquid hot water pretreatment (LHWP) at 180 °C for 20 min produced maximum xylose and glucose yields of 91.62% and 88.12%, respectively, which are higher than that of dilute acid pretreatment or individual LHWP. Corresponding concentration of acetic acid, HMF, and furfural in the prehydrolyzate are about 0.98, 0.07 and 0.78 g/L, respectively, which indicated that the detoxification methods are not required in next fermentation step. 90.7% of acid-insoluble lignin can be recovered in the enzymatic hydrolysis residue. It can be concluded that LHWP combined with WDM can be successfully applied to eucalyptus.
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Acknowledgements The authors would like to thank Genencor, Inc. (Jiangsu, China) for providing enzymes for this research. This research was supported financially by the Science and Technology Department of Guangdong Province, China (No. 2010y1-C071), the National Natural Science Foundation of China (No. 21176095), and the Major Research Projects of Guangdong Province, China (No. 2011A090200006).
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Hsu, T.C., Guo, G.L., Chen, W.H., 2010. Effect of dilute acid pretreatment of rice straw on structural properties and enzymatic hydrolysis. Bioresour. Technol. 101, 4907–4913. Inoue, H., Yano, S., Endo, T., Sasaki, T., Sawayama, S., 2008. Combining hotcompressed water and ball milling pretreatments to improve the efficiency of the enzymatic hydrolysis of eucalyptus. Biotechnol. Biofuels 1, 2. Jorgensen, H., Kristensen, J.B., Felby, C., 2007. Enzymatic conversion of lignocellulose into fermentable sugars: challenges and opportunities. Biofuels Bioprod. Biorefining 1, 119–134. Liu, C., Wyman, C.E., 2003. The effect of flow rate of compressed hot water on xylan, lignin, and total mass removal from corn stover. Ind. Eng. Chem. Res. 42, 5409– 5416. Liu, C., Wyman, C.E., 2004a. Impact of fluid velocity on hot water only pretreatment of corn stover in a flow through reactor. Appl. Biochem. Biotechnol. 113–116, 977–987. Liu, C., Wyman, C.E., 2004b. The effect of flow rate of very dilute sulfuric acid on xylan, lignin, and total mass removal from corn stover. Ind. Eng. Chem. Res. 86, 88–95. Liu, C., Wyman, C.E., 2005. Partial flow of compressed-hot water through corn stover to enhance hemicellulose sugar recovery and enzymatic digestibility of cellulose. Bioresour. Technol. 96, 1978–1985. Palmqvist, E., Hahn-Hägerdal, B., 2000. Fermentation of lignocellulosic hydrolysates. I: Inhibition and detoxification. Bioresour. Technol. 74, 17–24. Perez, J.A., Ballesteros, I., Ballesteros, M., Saez, F., Negro, M.J., Manzanares, P., 2008. Optimizing liquid hot water pretreatment conditions to enhance sugar recovery from wheat straw for fuel-ethanol production. Fuel 87, 3640–3647. Shuai, L., Yang, Q., Zhu, J.Y., Lu, F.C., Weimer, P.J., Ralph, J., Pan, X.J., 2010. Comparative study of SPORL and dilute-acid pretreatments of spruce for cellulosic ethanol production. Bioresour. Technol. 101, 3106–3114. Sun, Y., Cheng, J., 2002. Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresour. Technol. 83, 1–11. Vanderghem, C., Brostaux, Y., Jacquet, N., Blecker, C., Paquot, M., 2012. Optimization of formic/acetic acid delignification of Miscanthus giganteus for enzymatic hydrolysis using response surface methodology. Ind. Crops Prod. 35, 280–286. Wang, W., Yuan, T.Q., Wang, K., Cui, B.K., Dai, Y.C., 2012. Combination of biological pretreatment with liquid hot water pretreatment to enhance enzymatic hydrolysis of Populus tomentosa. Bioresour. Technol. 107, 282–286. Wei, W.Q., Wu, S.B., Liu, L.G., 2012. Enzymatic saccharification of dilute acid pretreated eucalyptus chips for fermentable sugar production. Bioresour. Technol. 110, 302–307. Yu, Q., Zhuang, X.S., Yuan, Z.H., Wang, Q., Qi, W., Wang, W., 2010. Two-step liquid hot water pretreatment of Eucalyptus grandis to enhance sugar recovery and enzymatic digestibility of cellulose. Bioresour. Technol. 101, 4895–4899. Zhu, W., Houtman, C.J., Zhu, J.Y., Gleisner, R., Chen, K.F., 2012. Quantitative predictions of bioconversion of aspen by dilute acid and SPORL pretreatments using a unified combined hydrolysis factor (CHF). Process Biochem. 47, 785– 791. Zhu, J.Y., Pan, X.J., 2010. Woody biomass pretreatment for cellulosic ethanol production: technology and energy consumption evaluation. Bioresour. Technol. 101, 4992–5002.
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Combination of liquid hot water pretreatment and wet disk milling to improve the efficiency of the enzymatic hydrolysis of eucalyptus Wei Weiqi a, Wu Shubin a,⇑, Liu Liguo b a b
State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, China Guangzhou You Rui Bio-Technology Limited Company, Guangzhou 510640, China
h i g h l i g h t s " Two environmentally friendly pretreatments were combined to enhance the sugar yield. " The concentration of fermentation inhibitors in the prehydrolyzate are discussed. " Compositions of water-insoluble solids resulting from pretreatment are discussed. " Find out the optimal pretreatment conditions basis on the total sugars yield. " A further analysis of insoluble solid resulting from enzymatic hydrolysis.
a r t i c l e
i n f o
Article history: Received 7 May 2012 Received in revised form 26 August 2012 Accepted 27 August 2012 Available online 7 September 2012 Keywords: Eucalyptus Liquid hot water pretreatment Wet disk milling Enzymatic hydrolysis Sugar recovery yield
a b s t r a c t Combination of liquid hot water pretreatment (LHWP) and wet disk milling (WDM) was investigated in this study to enhance the sugar recovery yield both in prehydrolyzate and enzymatic hydrolyzate. The results show that WDM with LHWP at 180 °C for 20 min produced maximum xylose and glucose yields of 91.62% and 88.12%, respectively, which are higher than that of dilute acid pretreatment or individual LHWP. Corresponding concentration of fermentation inhibitors such as acetic acid, HMF, and furfural in the prehydrolyzate are about 0.98, 0.07 and 0.78 g/L, respectively, which indicated that the detoxification may be not required in the next fermentation step. The acid-insoluble lignin recovery in the insoluble solid resulting from enzymatic hydrolysis, was 25.67/100 g raw material, representing 90.7% of acidinsoluble lignin in the eucalyptus biomass. It can be concluded that liquid hot water pretreatment combined with wet disk milling can be successfully applied to eucalyptus. Ó 2012 Published by Elsevier Ltd.
1. Introduction Due to a lack of resource sustainability of fossil fuels as well as negative environmental effects from emissions, research is being done to find a liquid fuel for use as a gasoline replacement (Arthur et al., 2011). Lignocellulosic materials are the most abundant biomass available to the world (Buranov and Mazza, 2008). The polysaccharides present in the lignocellulosic materials can be depolymerized to monosaccharide and then converted to ethanol via appropriate processes (Shuai et al., 2010). However, the natural structures of lignocellulosic materials make it hard for microorganism to utilize this material to produce ethanol. Therefore, efficient pretreatment method is needed so that sequential enzymatic hydrolysis gives maximal sugar productivity and at the same time any loss of sugar is minimized (Jorgensen et al., 2007).
⇑ Corresponding author. E-mail address: [email protected] (W. Shubin). 0960-8524/$ - see front matter Ó 2012 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.biortech.2012.08.130
Pretreatment techniques to overcome this recalcitrance are essential for saccharification and bioethanol production from lignocelluloses (Hendriks and Zeeman, 2008) in future biorefineries (Himmel et al., 2007). Various pretreatments using sulfuric acid, hydrochloric acid, formic acid and alkali reagents have been attempted (Cara et al., 2008; Vanderghem et al., 2012; Zhu and Pan, 2010). However, pretreatments using these chemicals have some problems such as the formation of inhibiting compounds for saccharification and subsequent ethanol fermentation and the pH requirements for downstream processes (Sun and Cheng, 2002). Moreover, the use of strong acids such as sulfuric acid in pretreatments has significant environmental risks. The development of environmentally friendly pretreatments that do not involve chemicals, such as ball milling (Endo et al., 2006), wet disk milling (Hideno et al., 2012) and liquid hot water pretreatment (Goh et al., 2010; Liu and Wyman, 2005), has been studied. However, they also have disadvantages. For example, ball milling has a high energy requirement and is not economically feasible in general (Inoue et al., 2008). Liquid hot water pretreatment,
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in which pressure is utilized to maintain water in the liquid state at elevated temperatures, has been reported to have the potential to remove most hemicelluloses, but the water-insoluble solid resulting from pretreatment are not very good for enzyme hydrolysis (Yu et al., 2010). To eliminate these disadvantages, Inoue et al. investigated ball milling combined with hot-compressed water treatment of eucalyptus, which can save energy for pretreatment and enzyme loading for enzymatic hydrolysis (Inoue et al., 2008). However, there have been very few reports about wet disk milling combined with liquid hot water pretreatment (LHWP), especially, for eucalyptus raw material. The objective of this work is to enhance the sugar recovery yield both in prehydrolyzate and enzymatic hydrolyzate through combination of liquid hot water pretreatment (LHWP) and wet disk milling (WDM). In addition, the compositions of water-insoluble solids resulting from liquid hot water pretreatment and the concentration of fermentation inhibitors such as acetic acid, HMF, and furfural in the prehydrolyzate are also discussed. Finally, there is a further analysis of insoluble solid resulting from enzymatic hydrolysis, found that most of the acid insoluble lignin can be recycled in the enzymatic hydrolysis residues. 2. Methods 2.1. Raw material The eucalyptus chips used in this study was provided by a local factory near LongYan, FuJian, China. The raw materials were airdried and then milled to give a size less than 5 mm by a Rub silk machine. The chemical composition of the raw material (on a dry weight basis) was 42.6% glucose, 15.4% xylose, 28.3% acid-insoluble lignin, 2.4% acid-soluble lignin, 3.3% extractives and 8.0% others. 2.2. Liquid hot water pretreatment Liquid hot water pretreatment was performed in a laboratory scale stirred autoclave. The reactor has a total volume of 1 L, with an electric heater and magnetic agitation. Temperature and agitation speed during the pretreatment was controlled and monitored with a modular controller (Perez et al., 2008). The amount of dry feedstock loaded was 75 g and water was added at 1/10 (w/v) solid/liquid ratio. Both water and raw material were initially at room temperature. Agitation was set at 500 rpm. The other conditions of pretreatment are summarized in Table 1. The pretreatment temperatures were designed within the range of 160–200 °C, with a residence time from 0 to 40 min. When the pretreatment was finished, the reactor was immediately cooled down by cooling water and then removed from heating jacket. The pretreated solution was then separated by filtration. The liquid fraction was analyzed by Table 1 Water-insoluble solids (WIS) fraction composition and recovery yield after liquid hot water pretreatment of eucalyptus under different pretreatment conditions. Conditions
Gravimetric recovery (%)
WIS composition (%)
20
87.28 82.48 77.66 74.24 71.35
44.83 46.91 51.38 53.41 53.96
0 10 20 30 40
80.87 78.29 77.66 76.89 75.39
45.35 49.47 52.98 53.61 54.48
Temp (°C)
Time (min)
160 170 180 190 200 180
Cellulose
HCel
AIL
ASL
Other
9.76 6.86 2.44 1.38 0.85
31.71 32.28 33.25 32.92 33.60
2.75 2.42 2.06 1.59 1.08
10.95 11.53 10.85 10.70 10.51
10.53 6.65 2.44 2.41 1.54
31.91 32.09 33.25 33.54 34.11
2.75 2.49 2.06 1.97 1.89
9.46 9.30 9.25 8.37 7.81
high-pressure liquid chromatography (HPLC) and ion chromatography (IC) to determine the concentration of inhibitors and sugars in the prehydrolyzate. The yield of water-insoluble solids (WIS) fraction was measured and then was stored in freezer for next step. 2.3. Wet disk milling pretreatment Wet disk milling was performed by a method based on previous reports (Endo et al., 2008; Hideno et al., 2009), using the Supermasscolloider MKZA10 (Masuko Sangyo Co., Ltd., Saitama, Japan) equipped with two ceramic nonporous disk grinders, which are adjustable for a clearance of 20–40 lm between the upper and lower grinders. The two disk grinders revolved at 1800 rpm. The liquid hot water pretreated residues were diluted with deionized water to 1/10 (w/v) solid/liquid ratio and then thrown into the machine, the operation was repeated 15 times. Slurry samples were taken after each operation and the moisture content of the samples was analyzed. 2.4. Enzymatic hydrolysis The enzymatic hydrolysis was then performed in 250 mL flasks using 50 mM sodium acetate buffer (pH 4.8) and 2% dry matter (w/ w) at 50 °C on an orbital shaker at 150 rpm for 60 h. The pretreated solid used for enzymatic hydrolysis was not extra handled by washing and drying process in this study. For preparing a 2% dry mass slurry, It first measured the moisture content of pretreated solid and then weighed the pretreated solid in wet basis which contained 2 g of dry mass. The pretreated solid weighed was further mixed with sodium acetate buffer with a final volume of 100 mL for enzymatic hydrolysis. The Cellulose enzyme was provided by Genencor, with a filter paper activity of 60 FPU/mL. The cellulase loading was set at 20 FPU/g dry matter. After the enzymatic hydrolysis test was completed, the sugars in enzyme hydrolyzate were analyzed by ion chromatography. 2.5. Analytical methods The chemical composition of raw material and water-insoluble solids (WIS) fraction resulting from liquid hot water pretreatment was determined by a standard analysis procedure of biomass composition, which was modified by the National Renewable Energy Laboratory (NREL) analytic methods (Cheng et al., 2006). All liquid samples from the pretreatment and enzymatic hydrolysis tests were filtered using a 0.45 lm filter and then diluted appropriately with the deionized water. The concentration of xylose, xylo-oligosaccharides, glucose and gluco-oligosaccharides were quantitatively analyzed by ion chromatography system (CarboPACTM PA1 column, ED detector, column temperature 30 °C, velocity 0.65 ml/min). The oligosaccharides in the liquid fraction were back-calculated after a secondary hydrolysis into monomer sugars with 4% sulfuric acid. The concentration of acetic acid, furfural and HMF were quantitatively analyzed at 45 °C using a HPLC system (Agilent 1200 series, Agilent Technologies) equipped with a refractive index detector. The sample separation was performed using a polystyrene–sulfonic acid column (Coregel-87H3 column, Transgenomic Technologies) at 65 °C with 4 mM H2SO4 as the eluent at a flow rate of 1.0 mL min 1 (Hsu et al., 2010). 3. Results and discussion 3.1. Compositions of WIS resulting from LHWP The total gravimetric recovery (solids remaining after liquid hot water pretreatment divided by original oven-dried weight) and the
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composition of water-insoluble solids resulting from liquid hot water pretreatment are summarized in Table 1. From the table, it can know that cellulose and acid-insoluble lignin is the major chemical composition of WIS, the content of hemicelluloses, ASL, and other material is relatively small. Part of the material was dissolved during liquid hot water pretreatment. As expected, with the rise of pretreatment temperature, the total gravimetric recovery is decreased. For example, when the pretreatment temperature is raised from 160 to 200 °C, corresponding gravimetric recoveries is decreased from 87.28% to 71.35%. In contrast, pretreatment time seems to exert a limited influence on total gravimetric recovery at a constant temperature. With the increase of pretreatment temperature or time, the cellulose content of WIS was increased. For example, when the pretreatment temperature is raised from 160 to 200 °C, corresponding cellulose content is increased from 44.83% to 53.96%. The cellulose content of WIS was always higher than the initial percentage of 42.6%, this is because the solubilization of water-soluble biomass components into prehydrolyzate resulted in a cellulose enrichment in WIS fraction. The acid-insoluble lignin content of WIS increased as pretreatment temperature or time increased, representing between 31% and 35% of the pretreated material. Referred to the initial content in raw material by considering the total gravimetric recovery, were 24–27%, which means that the pretreatment results in an acid-insoluble lignin degradation when compared to the original material (initial acid-insoluble lignin content in raw material 28.3%). Similar trends are reported in a study on the liquid hot water pretreatment with flowing hot water through cellulosic biomass in a small tubular flow through reactor by Liu and Wyman (2003, 2004a, 2004b). Moreover, interestingly, acid soluble lignin (ASL) demonstrated a decreased trend with the increase of pretreatment temperature or time, it is likely that the hot water solubilized part of the ASL, since hot water liberates acids and facilitates the breakage of such ether linkages in biomass during biomass hydrolysis (Wang et al., 2012). In addition, with the increase of pretreatment temperature or time, significant amounts of hemicellulose were degraded during liquid hot water pretreatment. For example, when the pretreatment temperature is above 200 °C, corresponding hemicellulose degradation is more than 97%, which implies that there was a nearly complete hydrolysis of hemicellulose during pretreatment under high severity conditions. The release of hemicallulose from the raw material during pretreatment can improve the efficiency of subsequent enzymatic hydrolysis (Zhu et al., 2012). 3.2. Prehydrolyzate composition Table 2 shows the composition of the prehydrolyzate obtained after liquid hot water pretreatment based on 100 g raw material. From the table, it can know that xylose and xylo-oligosaccharides
is the major carbohydrate in the prehydrolyzate liquor. The highest yield of total xylose in the prehydolyzate was 13.09 g (combined xylose and xylo-oligosaccharides, 3.39 and 9.70 g, respectively), representing 85% of total xylose in the eucalyptus biomass, which was generated after a pretreatment at 180 °C and 20 min residence time. Moreover, limited amounts of glucose and gluco-oligosaccharides were also released from cellulose during the liquid hot water pretreatment. The amount of total glucose (combined glucose and gluco-oligosaccharides) release was generally below 2.5 g per 100 g raw material. These results support the observation reported by others that the decomposition of hemicellulose starts at a lower temperature (180 °C) than that of cellulose in the liquid hot water. In addition, some furan byproducts from the conversion of sugars, such as furfural and HMF (hydroxymethylfurfural), were observed in the prehydrolyzate after pretreatment. Acetic acid was also detected in prehydrolyzate and believed to be generated from the release of the acetyl groups present in the hemicelluloses. These byproducts as mentioned above may have an inhibitory effect on the growth of xylose-fermenting microorganisms and reduce ethanol production. Fig. 1 shows the yield of sugars in the prehydrolyzate fractions as a function of pretreatment temperature or time. As expected, with the increase of pretreatment temperature or time, the yield of total xylose in prehydrolyzate is increase first, and then decrease. For example, when the pretreatment temperature is raised from 160 to 180 °C, corresponding yield of total xylose in prehydrolyzate is increased from 30.8% to 85.0%, continues to increase temperature, the yield of total xylose is quickly decreased. This decrease of total xylose in the prehydrolyzate can most likely be attributed to the degradation of xylose into furfural as a result of severe pretreatment conditions. The yield of xylo-oligosaccharides increased significantly during the first 20 min, at 180 °C, and then decreased quickly after about 20 min, which suggested that more xylo-oligosaccharides is hydrolyzed into xylose monomer for a long residence time or under more severe conditions. By contrast, the recovery yield of xylose monomer was increased continually from 4.03% to 26.36% with pretreatment residence time increased from 0 to 40 min, which suggested that the rate of xylo-oligosaccharides hydrolyzed into xylose monomer is much faster than that of xylose monomer hydrolyzed into furfural. Fig. 1 also shows that more xylose is oligomeric at a relatively low temperature. For example, the yield of xylo-oligosaccharides at 160 °C was 3.66 g, representing 77.22% of total xylose in the prehydrolyzate, and the yield of xylo-oligosaccharides at 200 °C was 4.23 g, representing 50.59% of total xylose in the prehydrolyzate. The yield of glucose and gluco-oligosaccharides in the liquid fractions was increased only slightly, which indicated that eucalyptus cellulose is recalcitrant to solubilization at these temperatures. Fig. 2 shows the yield of the fermentative inhibitors such as acetic acid, furfural and HMF at various pretreatment conditions. From
Table 2 Composition of the prehydrolyzate after liquid hot water pretreatment of eucalyptus under different pretreatment conditions. Conditions
Sugar analysis (g per 100 g raw material)
Inhibitor analysis (g/L)
Temp (°C)
Time (min)
Xylose monomer
Xylooligosaccharides
Total xylose
Glucose monomer
Glucooligosaccharides
Total glucose
Acetic acid
HMF
Furfural
160 170 180 190 200
20
1.08 2.02 3.39 4.03 4.13
3.66 6.36 9.70 5.14 4.23
4.74 8.38 13.09 9.17 8.36
nd 0.12 0.07 0.35 0.68
nd 0.36 0.63 1.00 1.65
nd 0.48 0.70 1.35 2.33
0.34 0.78 0.98 1.32 2.21
nd nd 0.07 0.17 044
0.24 0.44 0.78 2.61 3.29
180
0 10 20 30 40
0.62 1.23 3.39 3.45 4.06
4.26 9.85 9.70 7.92 5.51
4.88 11.08 13.09 11.37 9.57
nd nd 0.07 0.13 0.15
0.18 0.30 0.63 0.71 0.92
0.18 0.30 0.70 0.84 1.07
0.38 0.64 0.98 1.18 1.84
nd nd 0.07 0.09 0.08
0.12 0.42 0.78 1.66 2.05
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Yield of xylose / (%)
60
30 20
40
10
20 0
4
Residence time:20min 40
A
Yield of glucose / (%)
Xylo-oligosaccharides Xylose-monomer Total xylose Gluco-oligosaccharides Glucose-moomer Total glucose
80
0
160 170 180 190 Temperature of LHW pretreatment /
The concentration of inhibitors (g/L)
728
200
A Residence time:20min
3
Furfural 5-HMF Acetic acid
2
1
0 160
Temperature:180
60
30
40
20 Gluco-oligosaccharides Glucose-monomer Total glucose
20 0
10
B 10 30 0 20 Residence time of LHW pretreatment /
200
40
0 40
Fig. 1. Yields of sugars in the prehydrolyzate fraction resulting from different pretreatment conditions. (A) Pretreatment temperature and (B) pretreatment time.
Fig. 2, it can know that furfural which is generated by conversion from xylose is increased as the rise of both pretreatment temperature and time. For example, when the pretreatment temperature is raised from 160 to 200 °C, corresponding furfural content is increased from 0.24 to 3.29 g/L, which indicates that more xylose decomposition took place under severe conditions. Another important inhibitor, acetic acid, which is generated by the hydrolysis of the acetyl groups on hemicellulose is also increased with the strengthening of both pretreatment temperature and time. For example, when the pretreatment time is raised from 0 to 40 min, corresponding acetic acid content in the prehydrolyzate is increased from 0.38 to 1.84 g/L. In this study, when the yield of total xylose in the prehydolyzate was about 85%, corresponding concentrations of acetic acid, HMF, and furfural in the prehydrolyzate are about 0.98, 0.07 and 0.78 g/L, respectively. Previous studies have indicated that a decline in the ethanol yield from xylose fermentations when the concentration of acetic acid, HMF, and furfural was above 2.0, 1.0, and 1.0 g/L, respectively (Palmqvist and HahnHägerdal, 2000). Therefore, the detoxification methods, such as over-liming, vacuum evaporation and activated charcoal, may be not required to diminish the inhibition effect in next step of fermentation. 3.3. Enzymatic hydrolysis Table 3 shows the composition of the hydrolyzate obtained after enzymatic hydrolysis based on 100 g raw material. From the table it can know that glucose is the major carbohydrate in the enzyme hydrolyzate. With the increase of pretreatment temperature, there is a discernible upward trend in the yield of glucose. For example, when the pretreatment temperature is raised from 170 to 200 °C, corresponding glucose yield in enzymatic
The concentration of inhibitors (g/L)
Yield of xylose / (%)
80
Yield of glucose / (%)
Xylo-oligosaccharides Xylose-monomer Total xylose
190 180 170 Temperature of LHW pretreatment /
3
B Temperature:180 Furfural Acetic acid 5-HMF
2
1
0 0
30 20 10 Residence time of LHW pretreatment /
40
Fig. 2. Concentration of fermentation inhibitors in the prehydrolyzate resulting from different pretreatment conditions. (A) Pretreatment temperature and (B) pretreatment time.
Table 3 The yield of xylose, glucose and acid-insoluble lignin both in enzyme hydrolyzate and enzyme scraps under different pretreatment conditions (g per 100 g raw material). Optimal conditions
Enzyme hydrolyzate
Enzymatic hydrolysis residue
Xylose
Glucose
AIL
Xylose
Glucose
170 °C, 20 min 180 °C, 20 min 190 °C, 20 min 200 °C, 20 min 180 °C, 20 min + Wet disk milling
1.68 0.84 0.65 0.32 1.02
24.67 29.83 32.17 34.12 36.84
26.63 25.82 24.44 23.98 25.67
3.98 0.43 0.08 nd nd
14.02 10.07 7.48 4.38 3.09
hydrolyzate is increased from 24.67 to 34.12 g. Similar trends are reported in previous literature (Zhu et al., 2012). This upward trend can be explained by the fact that hemicellulose removal during the pretreatment in the form of either xylose or furfural can lead to high glucose yield in the hydrolyzate after the enzymatic hydrolysis. However, it was regretful to note that when the highest yield of total xylose in the prehydolyzate was about 85%, corresponding glucose yield in enzymatic hydrolyzate is only about 29.83 g (representing 70.02% of glucose in the eucalyptus biomass). Therefore, the post-pretreatment such as wet disk milling, are used to improve the reactivity of the water-insoluble solids with respect to enzymatic hydrolysis in this study. Related results are also summarized in Table 3, it was easy to find that the glucose yield in enzymatic hydrolyzate increased significantly when liquid hot water
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W. Weiqi et al. / Bioresource Technology 128 (2013) 725–730 Table 4 Yields of glucose and xylose from both liquid hot water pretreatment and enzymatic hydrolysis under optimal conditions (g per 100 g raw material).
c
Stage1a
Stage2b
Stage(1 + 2)c
Xylose
Glucose
Xylose
Glucose
Xylose
Glucose
170 °C, 20 min 180 °C, 20 min 190 °C, 20 min 200 °C, 20 min 180 °C, 20 min + Wet disk milling
8.38 13.09 9.17 8.36 13.09
0.48 0.70 1.35 2.33 0.70
1.68 0.84 0.65 0.32 1.02
24.67 29.83 32.17 34.12 36.84
10.06 13.93 9.82 8.68 14.11
25.15 31.18 33.52 36.45 37.54
Glucose and xylose in the prehydrolyzate. Glucose and xylose in the enzyme hydrolyzate. Glucose and xylose both in prehydrolyzate and enzyme hydrolyzate.
Xylose in prehydrolyzate The recovery yield of xylose / (%)
a b
Optimal conditions
100
Xylose in enzyme hydrolyzate
80
60
40
20
3.4. Total sugars Overall sugar yield, calculated in relation to the raw material for the two phases: pretreatment and enzymatic hydrolysis, is a major indicator of the potential amount of sugar that could be used for ethanol production. Therefore, in this study, the total sugars available for fermentation from eucalyptus are the summation of the xylose and glucose found in both the prehydrolyzate and the enzymatic hydrolyzate. The yields of glucose and xylose from both pretreatment and enzymatic hydrolysis are summarized in Table 4 and Fig. 3. From the table and figure, it can know that on the basis of 100 g raw material, the maximum xylose yield (combined xylose monomer and xylo-oligosaccharides) was about 14.11 g, representing 91.6% of total xylose in the eucalyptus biomass. This result compares favorably with that obtained when the same raw material was pretreated just with dilute sulfuric acid, where a maximum total xylose yield of 80.1% was attained (Wei et al., 2012). This improvement of xylose yield was attributed to the less degradation of xylose into furfural in the prehydrolyzate. On the basis of
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Glucose in prehydrolyzate The recovery yield of glucose / (%)
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in
0
17 0
pretreatment (LHWP) and wet disk milling (WDM) were combined compared with the individual liquid hot water pretreatment. For example, the glucose yield from the wet disk milled sample followed LHWP were 36.84 g (representing 86.48% of glucose in the eucalyptus biomass), which is obvious higher than that from the individual LHWP sample (29.83 g, representing 70.02% of glucose in the eucalyptus biomass). This increase of glucose yield after wet disk milling can most likely be attributed to the fiberization of cellulose by WDM. After the enzymatic hydrolysis, the insoluble solid resulting from enzymatic hydrolysis were treated with two step acid hydrolysis to test the remaining sugars and acid insoluble lignin (AIL) content. Related data were summarized in Table 3. It was easy to find an inverse proportion between the glucose yields from the enzymatic hydrolysis and the AIL content of the pretreated solid residue. For example, when the AIL content in pretreated solid residue is decreased from 26.63 to 23.98 g, corresponding yield of glucose in enzymatic hydrolyzate is increased from 24.67 to 34.12 g. This result implies that the lignin content of the pretreated solid residue is a critical factor to affect the performance of the enzymatic hydrolysis reaction in this study. Similar trends are reported in previous literature (Wei et al., 2012). Contrast the AIL yield in enzymatic hydrolysis residue, we can know that the lignin has not been degraded during the wet disk milling pretreatment. This is because the pretreatment of WDM is only a physical process, neither acids nor alkalis have been used in this process. Moreover, from Table 3, it can also know that, under the optimal pretreatment condition, the acid-insoluble lignin recovery in the enzymatic hydrolysis residue was about 25.67 g, representing 90% of acidinsoluble lignin in the eucalyptus biomass.
Fig. 3. The recovery yields of glucose and xylose from both pretreatment and enzymatic hydrolysis.
100 g raw material, the maximum glucose yield was about 37.54 g, representing 88.12% of total glucose in the eucalyptus biomass. This result compared with the individual liquid hot water pretreatment, where the highest glucose recovery yield was only about 73.19%, indicated that wet disk milling can greatly improve the reactivity of the water-insoluble solids with respect to enzymatic hydrolysis in this study.
4. Conclusions Wet disk milling (WDM) with liquid hot water pretreatment (LHWP) at 180 °C for 20 min produced maximum xylose and glucose yields of 91.62% and 88.12%, respectively, which are higher than that of dilute acid pretreatment or individual LHWP. Corresponding concentration of acetic acid, HMF, and furfural in the prehydrolyzate are about 0.98, 0.07 and 0.78 g/L, respectively, which indicated that the detoxification methods are not required in next fermentation step. 90.7% of acid-insoluble lignin can be recovered in the enzymatic hydrolysis residue. It can be concluded that LHWP combined with WDM can be successfully applied to eucalyptus.
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Acknowledgements The authors would like to thank Genencor, Inc. (Jiangsu, China) for providing enzymes for this research. This research was supported financially by the Science and Technology Department of Guangdong Province, China (No. 2010y1-C071), the National Natural Science Foundation of China (No. 21176095), and the Major Research Projects of Guangdong Province, China (No. 2011A090200006).
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