Bioresource Technology 193 (2015) 142–149

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Associating cooking additives with sodium hydroxide to pretreat bamboo residues for improving the enzymatic saccharification and monosaccharides production Caoxing Huang a, Juan He a, Yan Wang b, Douyong Min b, Qiang Yong a,⇑ a b

College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China College of Light Industry Science and Engineering, Nanjing Forestry University, Nanjing 210037, China

h i g h l i g h t s  Three pretreatments for sugars production were evaluated.  Alkali pretreatment was effective for bamboo saccharification.  Alkali pretreatment with cooking additive facilitated sugars releasing.

a r t i c l e

i n f o

Article history: Received 9 May 2015 Received in revised form 15 June 2015 Accepted 16 June 2015 Available online 24 June 2015 Keywords: Bamboo residues Pretreatment Cooking additive Enzymatic saccharification Monosaccharides

a b s t r a c t Cooking additive pulping technique is used in kraft mill to increase delignification degree and pulp yield. In this work, cooking additives were firstly applied in the sodium hydroxide pretreatment for improving the bioconversion of bamboo residues to monosaccharides. Meanwhile, steam explosion and sulfuric acid pretreatments were also carried out on the sample to compare their impacts on monosaccharides production. Results indicated that associating anthraquinone with sodium hydroxide pretreatment showed the best performance in improving the original carbohydrates recovery, delignification, enzymatic saccharification, and monosaccharides production. After consecutive pretreatment and enzymatic saccharification process, 347.49 g, 307.48 g, 142.93 g, and 87.15 g of monosaccharides were released from 1000 g dry bamboo residues pretreated by sodium hydroxide associating with anthraquinone, sodium hydroxide, steam explosion and sulfuric acid, respectively. The results suggested that associating cooking additive with sodium hydroxide is an effective pretreatment for bamboo residues to enhance enzymatic saccharification for monosaccharides production. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Lignocellulosic biomass, containing high polymeric carbohydrates, are regarded as renewable resources to obtain hexose and pentose for the production of bio-ethanol or bio-based chemicals (Herrera, 2004; Gusakov et al., 2007; Barr et al., 2012). Technically, bio-based products are usually produced from the sugar platform, which can be creased by monomeric sugars hydrolyzed from cellulose and hemicellulose (Barr et al., 2012). For decades, lignocellulose-based agriculture and forestry residues, such as sugarcane bagasse, wheat straw, and corn stover, have been extensively investigated to obtain the monosaccharides (glucose and xylose) (Jorgensen et al., 2007; Huang et al., 2015). Bamboo ⇑ Corresponding author. Tel./fax: +86 25 85427797. E-mail addresses: [email protected], [email protected] (Q. Yong). http://dx.doi.org/10.1016/j.biortech.2015.06.073 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.

is an economic forest and covers an area of 3.19 million hm2 in China. In bamboo utilization industry, fractions of bamboo green, bamboo yellow, bamboo node, and bamboo branches are discarded due to the weakness in mechanical properties, generating approximately 46 million tons processing residues annually (Chand et al., 2006). However, only a part of these bamboo residues are burned to recover energy in industry. Therefore, efficient utilization of bamboo residues is an ideal choice for bio-based chemical production and reducing the risk of pollution. In the cell wall of lignocellulosic biomass, cellulose is high-crystalline and associates with the hemicellulose and lignin, which are the major obstacles restricting for enzymes converting the polysaccharides into monosaccharides (Chen et al., 2011; Rahikainen et al., 2011). Therefore, an effective pretreatment is required to destroy the recalcitrant structure of lignocellulosic biomass and reduce the cellulose crystallinity for increasing the

C. Huang et al. / Bioresource Technology 193 (2015) 142–149

accessibility of cellulase to the cellulose (Mosier et al., 2005; Barr et al., 2012). For decades, the pretreatments of dilute sulfuric acid, steam explosion and sodium hydroxide have been investigated extensively. The dilute sulfuric acid pretreatment can degrade the hemicellulose, generating more pore for increasing accessibility portion of the biomass. Mosier et al. (2005) reported that high dilute sulfuric acid charge and high temperature can effectively degrade the hemicellulose and improve the enzymatic hydrolysis efficiency significantly. Steam explosion pretreatment has shown effective performance in degrading the lignin and hemicellulose, leading to an excellent enzymatic hydrolysis efficiency (Chen et al., 2014). Sodium hydroxide pretreatment can successfully disrupt the ester bonds between lignin and carbohydrates, resulting in the exposure of carbohydrates to enzymes (Li et al., 2010; Sambusiti et al., 2013). Meanwhile, alkali pretreatment provides the effective delignification and chemical swelling of the fibrous cellulose, which are the beneficial factors for achieving an excellent enzymatic saccharification efficiency (Zhao et al., 2009). Therefore, these pretreatments have been recognized as the effective methods to enhance the enzymatic digestibility of lignocellulosic biomass. Technically, an ideal pretreatment is able not only to produce a readily digestible substrate with low content of lignin, but also to recover the maximum amount of available sugars in feedstocks (Li et al., 2014). Due to the random alkali degradation and the peel reaction, a significant amount of carbohydrates are degraded during the alkali pretreatment process (Gu et al., 2012). Hence, an alkali-based pretreatment with maximizing the remained carbohydrates while minimizing the remained lignin in pretreated sample is considered as a potential method for monosaccharides production. In paper-making industry, cooking additives, such as sodium polysulphide (PS), anthraquinone (AQ) and borohydrate, have shown excellent performance in improving the degree of delignification and keeping as much carbohydrates as possible in the pretreated samples (Jameel et al., 1995; Copur and Tozluoglu, 2008). Although cooking additives have been extensively applied in paper-making industry for decades, applying these additives in the alkali pretreatment for the bioconversion of lignocellulosic biomass has not previously been reported. In this work, the monosaccharides (glucose and xylose) were obtained from bamboo residues after consecutive pretreatments and enzymatic saccharification process, which were pretreated by sulfuric acid, steam explosion and sodium hydroxide. Furthermore, cooking additives of sodium borohydride (NaBH4), sodium polysulphide (PS) and anthraquinone (AQ) were firstly applied in the sodium hydroxide pretreatment, with the goal of improving the monosaccharides production from the pretreated samples after enzymatic saccharification.

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of dry bamboo residues were put into the pot with certain volume of sulfuric acid solution. The sulfuric acid charge was varied in the range of 1–4% (w/v) and the ratio of solid to liquid was 1:10. The pretreated solids were washed with distilled water (with a dry mass-to-water ratio of 1:50) to neutrality. The resulting samples were stored at 4 °C for subsequent experiments. To elucidate the effect of acid pretreatment severity on carbohydrate recovery and delignification, the combined severity (CS) factor was used to characterization according to Chum et al. (1990), calculating as following equation:

R0 ¼ t  exp½ðT H  T R Þ=14:75 CS ¼ log R0  pH where R0 is severity factor, t is reaction time (min), TH is the target temperature in pretreatment (°C), TR is a reference temperature, and pH is the measured pH in initial cooking liquor. 2.2.2. Steam explosion pretreatment Steam explosion pretreatment was carried out in a batch pilot equipped with a 1 L reaction vessel. 100 g dry bamboo residues were packed into the reactor and maintained at the temperature of 180 °C, 200 °C and 220 °C for 10 min. After exposure at the designated condition, the bottom valve was opened to reduce the pressure to obtain the pretreated samples. The steam-exploded solids were washed with distilled water (with a dry mass-to-water ratio of 1:50) to neutrality. The resulting samples were stored at 4 °C for subsequent experiments. 2.2.3. Sodium hydroxide (NaOH) pretreatment Dry bamboo residues (100 g) were put into a 1 L cooking reactor with an oil bath. The pretreatment was performed at 120 °C, 140 °C and 160 °C for 60 min. The charge of NaOH was varied in the range of 2–10% (w/w) and the ratio of solid to liquid was 1:6. The pretreated solids were washed with distilled water (with a dry mass-to-water ratio of 1:50) to neutrality. The resulting samples were stored at 4 °C for subsequent experiments. 2.2.4. Sodium hydroxide pretreatment with cooking additives Before pretreatment, the cooking additive was added with sodium hydroxide (8%, w/w) into 1 L cooking reactor, charging with 100 g dry bamboo residues. The charges of NaBH4, PS, and AQ were 1–5% (w/w), 0.5–2.5% (w/w), and 0.03–0.15% (w/w), respectively. The pretreatments were maintained at 160 °C for 60 min. The pretreated solids were washed with distilled water (with a dry mass-to-water ratio of 1:50) to neutrality. The resulting samples were stored at 4 °C for subsequent experiments.

2. Methods

2.3. Enzymatic hydrolysis

2.1. Materials

Enzymatic hydrolysis experiment (50 mL) was performed at a substrate loading of 5% (w/v) and with an enzyme cocktail of 20 FPU/g-glucan cellulase and 3 IU/g-glucan b-glucosidase. The experiments were performed in a 150 mL Erlenmeyer flask at 50 °C using 50 mmol citrate buffer (pH4.8) with shaking at 150 rpm for 48 h. Aliquots were withdrawn and centrifuged for 10 min at 4000 rpm, the supernatants were subsequently filtered through a 0.22 lm syringe filter and analyzed for monosaccharides.

The residues used in this study were from the stems of older moso bamboo (Phyllostachys heterocycla) provided by the He Qi Cang Bamboo Processing Factory in Fujian, China. The primary chemical components of moso bamboo residues used in this work were as follows (%, dry weight basis): 39.21% glucan, 17.30% xylan, and 32.80% lignin. Cellulase (No. C2730, with an activity of 117 FPU/g) and b-glucosidase (No. NZ188, with an activity of 504 IU/g) were purchased from Sigma–Aldrich Inc. (USA).

2.4. Analysis methods 2.2. Pretreatment 2.2.1. Dilute sulfuric acid pretreatment Dilute sulfuric acid pretreatment was carried out in a 1 L cooking pot in an oil bath at 120 °C, 140 °C, and 160 °C for 60 min. 100 g

The chemical components of bamboo residues and pretreated samples were determined based on the procedure developed by the National Renewable Energy Laboratory for analyzing biomass materials.

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The concentration of monosaccharides in the supernatants were determined using a high performance liquid chromatography system with a refractive index (RI) detector. The separation was performed on an Aminex HPX-87H column (300  7.8 mm) with 5 mM H2SO4 as the eluent at a flow rate of 0.6 mL/min. The recovery yields of carbohydrates, degree of delignification, enzymatic hydrolysis efficiency and the amount of released monosaccharides (calculated on mass balance) were calculated according to the following equations:

Recovery yiled ð%Þ ¼

glucan or xylan in pretreated bamboo residueðgÞ  100% glucan or xylan in the raw bamboo residueðgÞ

Delignification ð%Þ ¼1

lignin in pretreated bamboo residueðgÞ  100% lignin in the raw bamboo residueðgÞ

Enzymatic efficiency ð%Þ ¼

glucose in enzymatic hydrolysateðgÞ  100% inital glucose in substrateðgÞ

Monosaccharide production ¼ released glucose and xylose in enzymatic hydrolysateðgÞ

3. Results and discussion 3.1. Dilute sulfuric acid pretreatment The corresponding acid pretreatment CS factors under different temperature and acid charge were calculated according to Chum et al. (1990) (Table 1). At 120 °C, the CS factor increased by 0.11– 0.36 with the increase of acid charge. Similar increase of pretreatment CS factor occurred at 140 °C and 160 °C. As shown in Table 1, increasing the pretreatment CS factor induced lower recovery yields of glucan and xylan in the pretreated samples. Most of the xylan in the bamboo residues was hydrolyzed when high pretreatment CS factor was employed. For instance, only glucan and lignin were left in the substrates pretreated by highest pretreatment severity with 1.71 CS factor (160 °C, 4%). A reason for the result is that cellulose in the lignocellulosic biomass is more stable than xylan to be hydrolyzed due to the crystalline nature and high

Table 1 Effects of sulfuric acid charge and pretreatment temperature on the recovery yield of carbohydrates and the degree of delignification (%). Temp. (°C)

Acid charge (w/v, %)

CS factora

Recovered yieldb Glucan

Xylan

120

1 2 3 4

0.17 0.28 0.37 0.53

97.25 ± 0.23 91.96 ± 0.53 89.43 ± 0.19 89.97 ± 0.28

49.19 ± 0.34 26.23 ± 0.83 23.65 ± 0.24 20.18 ± 0.53

10.15 ± 0.54 13.53 ± 0.62 15.37 ± 0.55 16.98 ± 0.23

140

1 2 3 4

0.76 0.87 0.96 1.12

92.45 ± 0.04 90.58 ± 0.70 87.37 ± 0.21 84.62 ± 0.43

17.37 ± 0.26 9.83 ± 0.13 4.77 ± 0.02 3.04 ± 0.25

12.13 ± 0.75 14.98 ± 0.26 16.03 ± 0.11 16.35 ± 0.53

160

1 2 3 4

1.34 1.45 1.54 1.71

89.26 ± 0.67 83.34 ± 0.73 83.57 ± 0.83 82.20 ± 0.36

11.06 ± 0.75 7.34 ± 0.11 0.00 ± 0.00 0.00 ± 0.00

14.37 ± 0.26 15.33 ± 0.38 16.26 ± 0.42 17.42 ± 0.29

Delignification

a Combined severity factor, coupling the reaction conditions of time, temperature, and acid charge into one single variable (Chum et al., 1990). b The proportion of remained carbohydrates in the pretreated bamboo residues, based on amount in the unpretreated bamboo residues.

degree of polymerization of the natural cellulose (Gu et al., 2013). In addition, it indicates that the increased pretreatment CS factor did not show an excellent performance in improving the delignification of bamboo residues, only 10.15–17.42% of the lignin was removed at a varied CS factor with 0.17–1.71. As shown in Fig. 1A, the increased pretreatment CS factor resulted in better enzymatic digestibility of the pretreated sample. For instance, an enzymatic hydrolysis efficiency with 4.62% was achieved from the sample pretreated by a pretreatment CS of 0.17. The highest pretreatment severity (CS factor = 1.71) resulted in the pretreated sample showed highest enzymatic hydrolysis efficiency of 24.59%. After statistical analysis (Fig. 1B), a liner correlation with y = 12.45x + 4.14 (r2 = 0.90) between the pretreatment severity and enzymatic saccharification efficiency was observed for the twelve pretreated samples. Similar correlation of pretreatment severity an digestibility has also been found in SO2-catalyzed steam exploded loblolly pine (Kang et al., 2013) and organosolv pretreated hardwood (Lai et al., 2014). They observed that higher acid-based pretreatment severity resulted in higher enzymatic digestibility of the pretreated sample. Unexpectedly, even though the hemicelluloses were almost dissolved during the dilute sulfuric acid pretreatment process, the enzymatic digestibility of the pretreated samples were still not as great. It may be explained that most lignin was retained in pretreated solid after pretreatment, the existing of remained lignin prevented the action of enzymes on polysaccharides by physical hindrance and unproductive enzyme binding (Kumar et al., 2012). From Fig. 1C, it is observed that the increased pretreatment severity induced more monosaccharides released from the pretreated samples after enzymatic saccharification. The highest amount of the released monosaccharides with 87.15 g was obtained from 1000 g of dry bamboo residues pretreated at 160 °C with 4% acid charge (CS factor = 1.71). 3.2. Steam explosion pretreatment In this work, steam explosion pretreatments with target temperature of 180 °C, 200 °C and 220 °C were applied to pretreat bamboo residues. Table 2 shows that an increased explosion temperature induced a reducing recovery yield of carbohydrates. After pretreatment, the recovery yields of glucan and xylan were 98.04% and 47.71%, respectively, at 180 °C; whereas the yields of glucan and xylan were 85.56% and 20.91%, respectively, at 220 °C. The decreased recovery yields of carbohydrates with the increased pretreatment temperature mainly attributed to the higher solubilization of carbohydrates under the prescribed pretreatment severity (Cara et al., 2006). Meanwhile, rising explosion temperature resulted in more removal lignin. For example, with the increased temperature from 180 °C to 220 °C, the degree of delignification was enhanced from 16.88% to 37.99%. It may be attributed to higher steam temperatures resulted in a deeper breakdown of lignocellulosic structure, thus facilitating the solubilization of lignin in the delignification process. It is worth noting that, at the lowest temperature (180 °C), steam explosion pretreatment did not effectively improve the enzymatic hydrolysis efficiency, with a yield only of 14.69%. With the increased explosion temperatures, considerable improvements in the susceptibility to enzymatic saccharification were evidenced. The enzymatic hydrolysis efficiency of the pretreated sample was enhanced from 14.69% to 35.04%, when the temperature rose from 180 °C to 220 °C. The improved enzymatic saccharification efficiency may be due to the different level of removal lignin and xylan in the pretreated samples (Yang et al., 2002; Zhang et al., 2013). Since lignin and xylan removal can break down the strong structure of cell wall, resulting in increasing the accessible pore surface to achieve excellent enzymatic saccharification of cellulose (Zhang

C. Huang et al. / Bioresource Technology 193 (2015) 142–149

(A)

Enzymatic efficiency (%)

bamboo residues, the enzymatic hydrolysis efficiency of the pretreated samples were still low. The maximum efficiency of enzymatic hydrolysis was only 35.04%. After the consecutive steam explosion pretreatment (220 °C) and enzymatic hydrolysis process, a maximum monosaccharides production with 142.93 g was achieved from 1000 g dry bamboo residues.

30 120 oC 140 oC 160 oC

25 20

3.3. Sodium hydroxide pretreatment

15 10 5 0 1

2

3

4

The concentration of sulfuric acid (w/v, %)

(B)

30

y=12.45x+4.14, r2=0.90

Enzymatic efficiency (%)

25 20 15 10 5 0 0.0

0.4

0.8

1.2

1.6

2.0

Combined severity factor (CS)

Released monosaccarides (g)

(C)

145

100 120 oC 140 oC 160 oC

80

60

40

20

0 1

2

3

4

The concentration of sulfuric acid (w/v, %) Fig. 1. The enzymatic hydrolysis efficiency (A), correlation between the CS factor and enzymatic efficiency (B), and released monosaccharides (C) of the bamboo residues pretreated by dilute sulfuric acid at different conditions. (The released monosaccharides (glucose and xylose) were calculated by mass balance based on 1000 g of dry bamboo residues, coupling pretreatment and enzymatic hydrolysis process.)

et al., 2013). Generally, it is believed that removal lignin from 20% to 65% is sufficient to increase the accessibility of cellulose to enzymes (Gu et al., 2013). In this work, although steam explosion pretreatment could remove one third of the original lignin from

Alkali-based pretreatments help to delignify, to disrupt the structure of lignocellulosic biomass, and to expose the cellulose to enzymatic attack producing high enzymatic saccharification efficiency (Gu et al., 2012; Li et al., 2014). As shown in Fig. 2A, with the increased alkali concentration and pretreatment temperature, the degree of delignification improved from 57.18% (2%, 120 °C) to 91.59% (10%, 160 °C). Compared to sulfuric acid and steam explosion pretreatments, it is found that NaOH pretreatment could effectively remove the lignin from bamboo residues. Wen et al. (2013) reported that bamboo lignin is an amorphous polymer not only consisting of p-hydroxyphenyl, guaiacyl and syringyl, but also containing some hydroxycinnamic acids (p-coumaric and ferulic acids) in the esterified and etherified forms. In the lignin macromolecule, a-aryl ether, arylglycerol-b-aryl ether, and ester linkage between lignin and hydroxycinnamic acids are more easily broken down at the alkali condition, which are beneficial for delignification (Wen et al., 2010). In Fig. 2B and C, the recovery yield of the carbohydrates decreased with the rising cooking temperature. For instant, at the condition of 8% NaOH concentration, the recovery yields of glucan and xylan decreased from 92.09% to 84.63% and from 34.05% to 27.70%, respectively, with a rising pretreatment temperature from 120 °C to 160 °C. It is also shown that the increased NaOH concentration induced a reducing recovery yield of carbohydrates. For example, the increased NaOH concentration from 2% to 10% resulted in the recovery yields of glucan decreased from 96.43% to 91.54% at 120 °C, 95.32% to 85.05% at 140 °C, and 93.67% to 80.21% at 160 °C, respectively. The intensified degradation of carbohydrates with increased alkali charge and pretreatment temperature may be attributed to severer random alkali degradation and peel reaction during pretreatment process (Gu et al., 2012). In addition, it is observed that the recovery yield of xylan was much lower than that of glucan in the sample pretreated by same condition. For example, bamboo residues pretreated by 8% NaOH at 160 °C, the recovery of glucan was 84.63%, while the yield of xylan was only 27.70%. Since hemicellulose has higher branched structures and more amorphous nature, the cellulose in bamboo residues was more stable than xylan to alkali exposure (Gu et al., 2013; Huang et al., 2015). When bamboo residues pretreated by NaOH, most of lignin and xylan were removed, which would make more pores for enzymes permeating into the cell wall to degrade the polymeric carbohydrates into monosaccharides. Fig. 3A shows that enzymatic hydrolysis efficiency improved with the rising cooking temperature and NaOH concentration. For instance, at condition of 2% NaOH and 120 °C, the enzymatic hydrolysis efficiency of the pretreated sample was only 22.41%, while the yield reached to 42.34% with the pretreatment condition of 2% NaOH and 160 °C. In addition, the highest enzymatic hydrolysis efficiency (81.43%) was achieved from the sample pretreated by 10% NaOH at 160 °C. Compered to the samples pretreated by sulfuric acid and steam explosion, the better enzymatic digestibility of the samples treated by sodium hydroxide may attributed to low level of lignin in the samples. Yu et al. (2011) indicated that the efficiency of enzymatic hydrolysis of lignocellulose was enhanced as the increased level of removal lignin. Meanwhile, the removal of lignin could reduce the strong surface interaction between the lignin and the enzyme (Lou et al., 2013). Xin et al. (2015) also found that alkali pretreatment

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Table 2 Effect of steam explosion pretreatment on the recovery yield of carbohydrates, delignification, enzymatic hydrolysis efficiency, and released monosaccharides. Temp. (°C)

Maintaining time (min)

Recovered yielda (%) Glucan

Xylan

180 200 220

10 10 10

98.04 ± 0.32 92.95 ± 0.22 85.56 ± 0.19

47.41 ± 0.31 31.13 ± 0.18 20.91 ± 0.13

Delignification (%)

Enzymatic hydrolysis efficiency (%)

Released monosaccharidesb (g)

16.88 ± 0.32 32.88 ± 0.26 37.99 ± 0.63

14.69 ± 0.33 28.04 ± 0.25 35.04 ± 0.63

70.89 ± 1.21 125.34 ± 2.73 142.77 ± 1.29

a

The proportion of remained carbohydrates in the pretreated bamboo residues, based on amount in the unpretreated bamboo residues. The monosaccharides (glucose and xylose) released were calculated by mass balance based on 1000 g of dry bamboo residues, coupling pretreatment and enzymatic hydrolysis processes. b

was shown the better performance in removing lignin and enhancing enzymatic hydrolysis efficiency of bamboo than dilute acid pretreatment. In addition, removal xylan in the pretreated sample was implicated in facilitating the biodegradation of lignocellulosic biomass besides the lignin. Zhang et al. (2013) reported significant xylan degradation is critical to increase the accessible area and pore for enzymatic cellulose saccharification. As shown in Fig. 3B, the maximum monosaccharides with 307.48 g was released from 1000 g dry bamboo residues, after the consecutive sodium hydroxide pretreatment (8%, 160 °C) and enzymatic hydrolysis process. However, increasing alkali charge from 8% to 10% failed to obtain more released monosaccharides from the pretreated sample. These phenomena may be attributed to more holocellulose losses at serve pretreatment conditions, which is not beneficial for sugars recovery (Monavari et al., 2009; Huang et al., 2015). Integrating all parameters, sodium hydroxide pretreatment with 8% alkali charge at 160 °C were the optimized conditions for monosaccharides (glucose and xylose) released from the bamboo residues. 3.4. Sodium hydroxide pretreatment with cooking additives As demonstrated above, the maximum monosaccharides production was achieved from bamboo residues pretreated by sodium hydroxide with 8% alkali charge at 160 °C. Although high enzymatic hydrolysis efficiency (76.17%) was achieved from this sample with a delignification degree of 89.73%, only 84.63% of original glucan and 27.70% of original xylan were retained in the sample. It is proposed that an ideal pretreatment is able not only to produce a readily digestible substrate with low content of lignin, but also to recover the maximum amount of available sugars in the feedstocks (Li et al., 2014). Hence, in order to maximize the remained carbohydrates while minimize the remained lignin in pretreated sample, cooking additives of sodium borohydride (NaBH4), sodium polysulphide (PS) and anthraquinone (AQ) were added during sodium hydroxide pretreatment process with 8% alkali charge at 160 °C. Table 3 demonstrates that associating these additives with sodium hydroxide resulted in pretreated samples with higher remained glucan and xylan, indicating that addition of these additives prevented carbohydrates degradation in alkali pretreatment. For instance, compare to the sample only pretreated by sodium hydroxide, increased recovery yields of glucan and xylan with 1.88% and 3.12% were achieved when 5% NaBH4 was added, which were from 84.63% to 86.51% for glucan and from 27.70% to 30.82% for xylan, respectively. Copur and Tozluoglu (2008) pointed out that NaBH4 is a powerful reducing agent that can convert the carbonyl group in the reducing end units of carbohydrate chains to hydroxyl groups, which is beneficial for prevention of peeling reaction of the carbohydrates. When 0.5–2.5% of PS was added with sodium hydroxide, the glucan and xylan recovery yields of the pretreated samples increased from 84.63% to 85.42–87.43% and from 27.70% to 27.82–30.01%, respectively. This phenomenon may be explained that PS can oxidize the aldehyde end groups in carbohydrates chains to aldonic acids during cleavage, stabilizing aldehyde

end groups against further peeing (Jiang, 1994). A significant improvement in glucan recovery was achieved when 0.03–0.15% of AQ was added in the alkali pretreatment. The increased yields may be explained that AQ could block peeling reactions by oxidizing polysaccharide end groups (Biswas et al., 2011). For xylan recovery yields, addition of AQ resulted in slightly lower recovery yields compared to sodium hydroxide pretreatment without cooking additives. In addition, it is observed that more lignin was removed from the samples pretreated by sodium hydroxide with these cooking additives. Maximum increases of delignification degree were achieved from the samples pretreated by sodium hydroxide with 5% NaBH4, 2.5% PS and 0.15% AQ, which were from 89.73% to 91.89%, 93.01% and 95.55%, respectively. Table 3 also shows that enzymatic digestibility of the pretreated sample was correlated with the cooking additives charge. Increasing the charge of NaBH4 from 0% to 5%, the enzymatic hydrolysis efficiency gradual improved, which was from 76.17% to 80.93%. The enzymatic hydrolysis efficiency also enhanced with the increased charge of PS. Somewhat surprising, highest improvement of enzymatic hydrolysis efficiency was achieved from the sample pretreated by sodium hydroxide with 0.15% AQ, with a yield of 82.03%. The increased enzymatic saccharification efficiency may be induced by more removal lignin in the samples pretreated with cooking additives. Since lignin coverage over cellulose fibrils plays a critical role in protecting carbohydrates from degradation (Zhang et al., 2013). Removing lignin from biomass can increase substrate accessibility for the enzymatic saccharification of polysaccharides (Yang and Wyman, 2004). These results suggested that adding cooking additives during the sodium hydroxide pretreatment process played an important role in improving the enzymatic saccharification efficiency of pretreated samples. As expected, the released monosaccharides was enhanced with the increased charge of cooking additives. As observed in the Table 3, increasing NaBH4 from 0% to 5% resulted in an increase of monosaccharides from 307.48 g to 336.51 g (based on 1000 g of dry bamboo residues). A similar tendency was also achieved from the samples pretreated by sodium hydroxide with PS. After enzymatic saccharification, the highest monosaccharides with 347.49 g was released from 1000 g of dry bamboo residues pretreated by sodium hydroxide with 0.15% AQ. Compare to sample pretreated by sodium hydroxide, more monosaccharides was released from the sample pretreated by sodium hydroxide with cooing additives. It may be due to the higher enzymatic saccharification efficiency and more remained original carbohydrates in these samples. Integrating all cooking additives with different charge, sodium hydroxide pretreatment with 0.15% AQ showed best performance in improving the monosaccharides production from pretreated bamboo residues. 3.5. Comparison of the pretreatments Compared to sodium hydroxide pretreatment, dilute sulfuric acid and steam explosion pretreatment had very weak ability to remove the lignin from bamboo residues. For example, at the

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(A)

(A)

100

100

o

Degree of delignification (%)

90

Enzymatic efficiency (%)

120 C 140 oC 160 oC

80 70 60 50

80

60

40 120 oC 140 oC 160 oC

20

40 0

0

2

4

6

8

10

2

NaOH concentration (w/w, %)

(B)

100

120 oC 140 oC 160 oC

Recovery yield of glucan (%)

95 90 85 80 75 70 0 2

4

6

8

10

NaOH concentration (w/w, %)

120 oC 140 oC 160 oC

80

60

40

20

0 2

4

6

8

6

8

10

350 300 250 200 160 oC 140 oC 120 oC

150 100 0 2

4

6

8

10

NaOH concentration (w/w, %)

100

Recovery yield of xylan (%)

(C)

Released monosaccarides (g)

(B)

4

NaOH concentration (w/w, %)

10

NaOH concentration (w/w, %) Fig. 2. The degree of delignification (A), recovery yields of glucan (B) and xylan (C) of the bamboo residues pretreated by sodium hydroxide at different conditions. (The recovery carbohydrates refer to the remained carbohydrates in the pretreated bamboo residues.)

prescribed pretreatment condition, the maximum delignification degree of sample pretreated by sodium hydroxide reached to 91.59%, while that of samples pretreated by dilute sulfuric acid and steam explosion only were 17.42% and 41.38%, respectively. The enzymatic saccharification efficiencies of these pretreated samples were 81.43%, 24.59%, and 31.40%, respectively. After liner

Fig. 3. The enzymatic hydrolysis efficiency (A) and released monosaccharides (B) of the bamboo residues pretreated by sodium hydroxide at different conditions. (The released monosaccharides (glucose and xylose) were calculated by mass balance based on 1000 g of dry bamboo residues, coupling pretreatment and enzymatic hydrolysis process.)

fitting, a very strong correlation (y = 0.801x + 5.621, r2 = 0.96) between the degree of delignification and enzymatic saccharification efficiency was observed for these pretreated samples. The results imply that degree of delignification is a key factor affecting the enzymatic digestibility of bamboo residues. Since the existing of lignin prevents the action of enzyme on polysaccharides by physical hindrance and unproductive enzyme binding (Kumar et al., 2012). After the consecutive pretreatment and enzymatic hydrolysis process, 307.48 g, 142.93 g and 87.15 g (based on 1000 g of dry bamboo residues) of monosaccharides were released from samples pretreated by sodium hydroxide, steam explosion, and sulfuric acid, respectively. This result is in agreement with Xin et al. (2015) that alkali-based pretreatment is a more effective method for the bioconversion of bamboo residues to monosaccharides than acid-based pretreatment. When the cooking additives (NaBH4, PS, and AQ) were added in the sodium hydroxide pretreatment process, AQ showed best performance in improving the carbohydrates recovery, delignification, enzymatic saccharification, and monosaccharides released. A maximum amount of monosaccharides with 347.49 g was released from 1000 g dry bamboo residues after the consecutive pretreatment of sodium hydroxide with 0.15% AQ and enzymatic saccharification process. These results suggest that sodium hydroxide associating

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Table 3 Effects of three cooking additives on the improvement for carbohydrates recovery yield, delignification, enzymatic hydrolysis efficiency, and released monosaccharides. Additives

Charge (%, w/w)

Recovered yielda (%) Glucan

Xylan

Nonec Sodium borohydride (NaBH4)

0 1 2 3 4 5

84.63 ± 0.23 85.22 ± 0.21 85.98 ± 0.11 86.45 ± 0.45 86.31 ± 0.25 86.51 ± 0.37

Sodium polysulphide (PS)

0.5 1.0 1.5 2.0 2.5

Anthraquinone (AQ)

0.03 0.06 0.09 0.12 0.15

Delignification (%)

Enzymatic hydrolysis efficiency (%)

Released monosaccharidesb (g)

27.70 ± 0.17 28.01 ± 0.23 28.97 ± 0.04 29.54 ± 0.63 30.11 ± 0.23 30.82 ± 0.02

89.73 ± 0.87 89.99 ± 0.51 90.34 ± 0.89 90.89 ± 0.55 91.03 ± 0.67 91.89 ± 0.83

76.17 ± 0.01 77.21 ± 0.11 78.41 ± 0.01 79.08 ± 0.06 80.22 ± 0.24 80.93 ± 0.02

307.48 ± 1.31 314.05 ± 2.01 322.30 ± 0.42 327.35 ± 2.21 332.03 ± 1.22 336.51 ± 1.41

85.42 ± 0.07 86.21 ± 0.81 86.69 ± 0.19 87.51 ± 0.42 87.43 ± 0.74

27.82 ± 0.04 28.45 ± 0.66 29.00 ± 0.22 29.45 ± 0.23 30.01 ± 0.71

90.21 ± 0.82 90.81 ± 0.25 91.63 ± 0.43 92.41 ± 0.42 93.01 ± 0.11

77.23 ± 0.14 78.22 ± 0.31 79.41 ± 0.15 80.35 ± 0.22 81.21 ± 0.30

314.75 ± 0.12 322.13 ± 2.21 329.10 ± 1.21 336.51 ± 2.01 340.81 ± 3.47

85.71 ± 0.21 86.21 ± 0.01 87.52 ± 0.17 88.49 ± 0.36 89.33 ± 0.33

27.91 ± 0.24 28.24 ± 0.11 27.21 ± 0.71 26.93 ± 0.26 26.53 ± 0.18

90.64 ± 0.42 91.34 ± 0.26 92.74 ± 0.27 93.94 ± 0.34 95.55 ± 0.36

77.83 ± 0.03 78.56 ± 0.07 79.29 ± 0.42 80.54 ± 0.32 82.03 ± 0.22

318.31 ± 0.83 323.43 ± 0.21 329.94 ± 0.91 338.09 ± 1.43 347.49 ± 1.22

a

The proportion of remained carbohydrates in the pretreated bamboo residues, based on amount in the unpretreated bamboo residues. The monosaccharides (glucose and xylose) released were calculated by mass balance based on 1000 g of dry bamboo residues, coupling pretreatment and enzymatic hydrolysis processes. c The pretreatment was with 8% alkali charge at 160 °C without cooking additive. b

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