Bioresource Technology 149 (2013) 375–382

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Effects of green liquor pretreatment on the chemical composition and enzymatic digestibility of rice straw Feng Gu 1, Wangxia Wang 1, Lei Jing, Yongcan Jin ⇑ Jiangsu Provincial Key Lab of Pulp and Paper Science and Technology, Nanjing Forestry University, Nanjing 210037, China

h i g h l i g h t s  Green liquor (GL) was used as an effective pretreatment on rice straw.  Most polysaccharides retained in pretreated solid with a high delignification.  Most silica was kept in the residue as a potential high value-added byproduct.  GL pretreatment can significantly improve the efficiently of enzymatic hydrolysis.  The maximum sugar yield after GL pretreated and enzymatic hydrolysis was 78%.

a r t i c l e

i n f o

Article history: Received 4 July 2013 Received in revised form 13 September 2013 Accepted 17 September 2013 Available online 27 September 2013 Keywords: Rice straw Green liquor pretreatment Delignification Enzymatic digestibility Sugar yield

a b s t r a c t Green liquor (Na2S + Na2CO3, GL) pretreatment is a proven pathway to improve the enzymatic saccharification for the production of bioethanol. In this work, the effects of GL pretreatment on the chemical composition and enzymatic digestibility of rice straw at various total titratable alkali (TTA) charge and temperature were investigated. The GL pretreatment showed excellent performance in high polysaccharides retention and delignification selectivity. Under the optimized GL pretreatment condition (4% TTA charge, 20% sulfidity and 140 °C), 92.5% of glucan, 82.4% of xylan and 81.6% of arabinan in rice straw were recovered with a delignification of 39.4%. The maximum sugar yields of 83.9%, 69.6% and 78.0%, respectively for glucan, xylan and total sugar, were achieved at the same GL pretreatment condition with an enzyme loading of 40 FPU/g-substrate. The results suggested that GL pretreatment is a practicable method for rice straw to enhance enzymatic saccharification for bioethanol production. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Fossil fuel, including petroleum, coal, and natural gas, is the most popular energy resource. Over 85% of the energy demands are met by the combustion of fossil fuels, and it is estimated that these non-renewable resources will be used out in the next 40–150 years (Shafiee and Topal, 2009). Meanwhile, as a result of fossil fuel consumption, CO2, NOx, SOx and other pollutants will cause considerable damage to the environment. Exploiting the renewable resources to replace the depleting energy is the only way to realize the sustainable development of society. There are various new energy resources on the earth, such as energy from the sun, the wind, the water and the biomass. Compared with other

⇑ Corresponding author. Address: Laboratory of Wood Chemistry, Department of Paper Science and Technology, Nanjing Forestry University, 159 Longpan Rd., Nanjing 210037, China. Tel.: +86 (25) 8542 8163; fax: +86 (25) 8542 8689. E-mail addresses: [email protected] (F. Gu), [email protected] (W. Wang), [email protected] (L. Jing), [email protected] (Y. Jin). 1 These authors contributed equally to this work. 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.09.064

alternative energy, biomass resources can be converted into liquid fuel which providing the most convenient storage mode (Szczodrak and Fiedurek, 1996). So the utilization of biomass as a solution to substitute fossil fuel has attracted much attention. Rice straw (Oryza sativa L.) offers great potential as a raw material for bioethanol production. According to FAO (Food and Agriculture Organization of the United Nations) statistics, the production of rice straw is about 650–975 million tons per year globally, which is calculated according to the ratio of straw to grain (Binod et al., 2010). Until now, the rice straw has not been used reasonably or just burned in field resulting air pollution and greenhouse gas emission. Efficient utilization of rice straw resource is the best choice for both sides of providing bioenergy and releasing risk of environmental pollution (Chen et al., 2009). Cellulose, hemicellulose and lignin are the major cell wall components in lignocellulosic materials. Rice straw containing 50–80% carbohydrates is viewed as a potential alternative to our current reliance on fossil fuels (Saha, 2003). However, cellulose is high-crystalline and closely associated with hemicellulose and lignin, which is the major obstacle restricting polysaccharides

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degradation by hydrolytic enzymes, thereby limiting the bioconversion of lignocellulosic materials into liquid fuels (Rahikainen et al., 2011). Therefore, development of practical and environmentally friendly pretreatment methods for reducing cellulose crystallinity, disrupting the association and partially removing lignin and hemicellulose is of great importance to bioethanol production from lignocellulosic biomass. Pretreatment is an essential step towards the development and industrialization of efficient 2nd generation lignocellulosic ethanol processes (Chiaramonti et al., 2012). However, it has been recognized as a technological bottleneck for the cost-effective development of bioprocess from biomass (Mosier et al., 2005). Green liquor (GL) pretreatment on woody biomass has shown effective performance in improving enzymatic digestibility (Jin et al., 2010; Wu et al., 2010). All the equipment and processes in GL pretreatment system have been industrially practiced for many decades in dozens of kraft pulp mills in the world, and the chemicals of green liquor, sodium carbonate and sodium sulfide, can be completely recovered from a proven chemical recovery system. Meanwhile, GL pretreatment keeps as much polysaccharides as possible in the substrate for enzymatic hydrolysis, and all fermentable sugar can be recovered in one step. It avoids fermentable sugars collection from both pretreatment and enzymatic hydrolysis steps such as steam explosion pretreatment, and retains a higher sugar concentration for fermentation to ethanol. An important benefit of GL pretreatment is that no toxic byproducts such as furfural, acetic acid (from hemicellulose degradation) are produced to affect the fermentation step and cause corrosion in the equipment. An interest possibility that may reduce the process cost is to integrate ethanol production with a pulp mill or to repurpose a kraft pulp mill to a bioethanol plant. Considering the capital costs, investment risk, technical feasibility, especially the efficiency for bioethanol production, the GL process provides an effective pretreatment method for bioethanol production from lignocellulosic biomass. In this study, rice straw was pretreated by green liquor at various total titratable alkali (TTA) charge and temperature, to understand the effects of GL pretreatment on the chemical composition and enzymatic hydrolysis of rice straw for the production of bioethanol or sugar based chemicals. 2. Methods 2.1. Materials Rice straw used as feedstock in this work was collected in Jiangsu, China. Air dried raw materials without classification, including stem, leaf and sheath was cut into a length of 3–5 cm and stored in sealed plastic bags at 4 °C in a refrigerator. Prior to composition analysis, the biomass was ground using a Wiley mill, and the particles between 40 and 80 mesh were collected. All weights and calculations were made on the basis of oven dry materials (DM). Three enzymes, cellulase from Trichoderma reesei (NS-50013, 52.3 mg protein/mL, 84 FPU/mL), b-glucosidase from Aspergillus niger (NS-50010, 48.5 mg protein/mL, 350 CBU/mL) and xylanase (NS-50014, 50.2 mg protein/mL, 850 FXU/mL) were provided by Novozymes (Franklinton, NC, USA). All the chemicals used for pretreatment and enzymatic hydrolysis were analytical grade and purchased from Nanjing Chemical Reagent Co., Ltd. of China and used as received without further purification. 2.2. Green liquor pretreatment Green liquor solution was prepared by mixing Na2S and Na2CO3 with a sulfidity of 20%. The definition of sulfidity is the ratio of

Na2S–Na2S and Na2CO3 (on Na2O basis). The TTA charge as Na2O on oven dry material was 0–16%. GL pretreatment was carried out in a ten-bomb (1 L) lab scale pulping system with oil bath. The ratio of pretreatment liquor to biomass was 6 (v/w). The raw materials were first impregnated with the pretreatment liquor at 60 °C for 30 min. After impregnation, the temperature was raised with the rate of 2 °C/min to the target temperature (100–160 °C) and maintained for 1 h. The pretreatment was terminated immediately by cooling the bombs to room temperature in cold water. The pretreated solid was collected and washed with water to remove residual chemicals and dissolved straw components. Solid recovery was calculated according to the wet weight and moisture content of the collected solid. The pretreatment spent liquor was collected for pH analysis. 2.3. Enzymatic hydrolysis A laboratory refiner (U 300 mm, 3000 rpm, KRK, Jilin, China) was used to defiberize the pretreated solids to prepare substrates for enzymatic hydrolysis. Enzymatic hydrolysis of the substrates was carried out in a 150 mL Erlenmeyer flask at a consistency of 5% (w/w) in sodium acetate buffer (0.2 M, pH = 4.8) at 50 °C using a shaking incubator (DHZ-2102, Jinhong, Shanghai, China) at 180 rpm for 48 h. An enzyme cocktail mixed by cellulase (NS-50013), xylanase (NS-50044) and b-glucosidase (NS-50010) was used for the enzymatic hydrolysis of GL pretreated samples. The dosages of b-glucosidase and xylanase supplementation constituted 30% of the volume of cellulase added, according to the suggestion from the manufacturer. The enzyme loadings were 5, 10, 20 and 40 filter paper units (FPU) per gram of substrate based on cellulase activity. Sodium azide was charged at 0.3% (w/v), based on total volume of the pulp slurry as an antibiotic to inhibit microbial growth during the enzymatic hydrolysis. Enzymatic hydrolysis residue and hydrolysate was separated by centrifugation after boiled in water for 5 min. Hydrolysate was sampled for monomeric sugar (glucose, xylose and arabinose) analysis. Each data point was the average of duplicate experiments. 2.4. Analytical methods The enzymatic hydrolysate was diluted 2000 times with the addition of L-()-fucose (F2252, Sigma, Saint Louis, MO, USA) as internal standard. Monomeric sugars were determined using an improved high performance anion exchange chromatography (ICS-3000, Dionex Corp., Sunnyvale, CA, USA) with pulsed amperometric detector (HPAEC-PAD). A CarboPac™ PA1 (2  250 mm) and a CarboPac™ PA1 (2  50 mm) (Dionex Corp., Sunnyvale, CA, USA) were used as analytical and guard column. An 18 mmol/L NaOH solution prepared with degassed super-purified deionized water was used as eluent at a flow rate of 0.25 mL/min. Aliquots (5 lL) were injected after passing through a 0.22 lm nylon syringe filter. The column was reconditioned by using 200 mmol/L NaOH after each three analysis. Monomeric sugars were quantified with reference to standards using the same analytical procedure. The concentration of monosaccharide was corrected by calibration curve of standard sugars. The average of duplicate runs was used in reporting. Data of monomeric sugar content were corrected to polymeric sugar for yield calculation. The contents of hot water, 1% NaOH and benzene–ethanol extractives were determined according to Tappi Standard T207 cm-99, T212 om-98 and T204 cm-97, respectively. Lignin and carbohydrates of raw materials and pretreated solids were analyzed by Laboratory Analytical Procedures from National Renewable Energy Laboratory (NREL) (Sluiter et al., 2008). The Klason lignin (KL) content was taken as the ash free residue after acid hydrolysis. The hydrolysate from this determination was

F. Gu et al. / Bioresource Technology 149 (2013) 375–382

retained for the analysis of sugars and acid soluble lignin (ASL). Sugars were determined as described earlier, except that sugar standards were autoclaved at 121 °C for 1.0 h prior to analysis to compensate for destruction during heating. Acid soluble lignin was analyzed by absorbance at 205 nm in a UV–vis (TU-1810, Puxi, Beijing, China) spectrometer. The contents of ash and silica were determined according to the methods described by Pan et al. (1999). Cellulase activity was determined by the filter paper method using Whatman No. 1 filter paper as a standard substrate (Ghose, 1987).

3. Results and discussion 3.1. Characterization of rice straw The purpose of pretreatment is to change raw material properties, remove or dissolve part of lignin and hemicellulose, and reduce cellulose crystalline (Kumar et al., 2009). The optimal pretreatment condition usually mainly depends on the species of lignocellulosic materials. Herbaceous plant generally consists of stem and leaves. The biological structure of leaves in herbaceous plant is different with that of stem. Leaves contain much non-fiber components, such as parenchyma, which are suggested to be separated out during pulp and paper making process to eliminate the adverse effect on paper quality (Kolmer et al., 2007). However, the polysaccharides in both leaves and stem of non-wood fiber materials can be hydrolyzed into fermentable sugar, which can be used for bioethanol production. The main chemical components of rice straw used in this work, including reducing sugars, are given in Table 1. Compared with wood materials, straw fiber materials are characterized by lower lignin and higher pentosan or hemicellulose contents (Munawar et al., 2007). The total lignin content of rice straw was 19.9%, which containing higher percentage of acid soluble lignin (15%) than most wood materials. The total sugar content was 53.9%, in which glucan (cellulose) and pentosan (hemicellulose) taken up 63% and 37%, respectively. Another major feature of rice straw is the high ash content and about two thirds of that is silica. It should be noted that the content of 1% NaOH extractives was as high as 49.6%, which implies that the chemical structure of rice straw is flexible to be disrupted under weak base condition. A possible explanation for this phenomenon is that the ester linkages between lignin and hemicellulose via ferulic and coumaric acid is quite flexible under alkaline condition in the Gramineae (Buranov and Mazza, 2008).

377

3.2. Solid recovery and spent liquor pH of GL pretreated rice straw The ethanol production yield from natural lignocellulosic biomass is relatively low due to its native recalcitrance, which is mainly attributed to many substrate-related physical and chemical parameters, such as lignin content, hemicellulose content and the cross-linking between these major components (Leu and Zhu, 2013). Pretreatment of lignocellulosic materials is required to overcome this recalcitrance by altering the physical features and chemical composition/structure of lignocellulosic materials, to make cellulose more accessible to enzymatic hydrolysis for sugar conversion (Hu and Ragauskas, 2012). In this work, rice straw was pretreated using simulated green liquor with TTA charge (as Na2O) varying from 0% to 16%. The simulated green liquor was prepared in laboratory by mixing sodium carbonate and sodium sulfide with a sulfidity of 20%. The pretreatments were carried out under the conditions described earlier. The solid recovery and spent liquor pH of GL pretreatment are given in Figs. 1 and 2, respectively. The pretreated solid recovery was calculated by Eq. (1):

Pretreated solid recovery ð%Þ ¼

Dry pretreated solid ðgÞ  100% Dry raw material ðgÞ ð1Þ

In GL pretreatment, an obvious drop of pulp yield was observed with the increase of TTA charge and pretreatment temperature, especially for TTA charge 0–8% and temperature 120–160 °C. The descending pulp yield mainly caused by the degradation of hemicellulose and lignin, as well as some cellulose. Lignin is generally recognized as an obstacle restricting polysaccharides degradation, so partial lignin removal is benefit for improving sugar yield in subsequent hydrolysis (Han et al., 1983). An ideal pretreatment should reduce the loss of fermentable sugar in the process for a high efficiency of enzymatic hydrolysis (Jørgensen et al., 2007). However, due to the interaction between lignin and carbohydrates, a part of cellulose and hemicellulose were inevitably degraded from rice straw after GL pretreatment, especially under severe conditions. Though the removal of hemicellulose is beneficial to improve the enzymatic digestibility of the pretreated substrate, the final sugar yield may be reduced if too much polysaccharide is lost in alkaline condition. The degradation of hemicellulose in alkaline media produced some acid compounds, such as acetic acid and uronic acid. It resulted in a lower pH-value of spent liquor, as shown in Fig. 2. It

Table 1 The main chemical composition of rice straw (standard deviations are given between brackets). Component

Content (%)

Extractives Benzene-ethanol 1% NaOH Hot water

3.7 (0.4) 49.6 (0.3) 17.2 (0.4)

Lignin Klason lignin Acid soluble lignin

19.9 (0.1) 16.9 (0.1) 3.0 (0.0)

Sugar Glucan Xylan Arabinan

53.9 (0.7) 33.7 (0.4) 17.4 (0.2) 2.8 (0.2)

Ash SiO2

12.4 (0.2) 8.3 (0.1)

Fig. 1. Solid recovery of rice straw as a function of TTA charge in GL pretreatment at different temperature.

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Sugar recovery ð%Þ ¼

Sugar in pretreated solid ðgÞ  100% Sugar in raw material ðgÞ

ð2Þ

Lignin in raw material ðgÞ  Lignin in pretreated solid ðgÞ Lignin in raw material ðgÞ  100% ð3Þ

Lignin removal ð%Þ ¼

Silica retention ð%Þ ¼

Fig. 2. Spent liquor pH as a function of TTA charge in GL pretreatment at different temperature.

is clearly that the pH-value drops with the increase of pretreatment temperature at same TTA charge, since more acid compounds are released into the liquor at higher temperature. All the pHvalues of the pretreatment spent liquor ranged from 7.8 to 10.3, which were considerably lower than that of a normal kraft pulping (13–14). This low final pH is an important feature of the GL pretreatment, especially for straw fiber materials. Low pH prevents the alkaline hydrolysis and secondary peeling reactions of polysaccharide from occurring during the pretreatment, which results in higher recovery of polysaccharide in pretreated solid (Sjöström, 1993). Meanwhile, most silica was kept in the pretreated solid rather than dissolved in the spent liquor at a weak alkaline condition. It is beneficial to eliminate the problems of spent liquor evaporation and combustion in chemical recovery caused by the dissolved silicate. 3.3. Chemical composition of GL pretreated rice straw The content of various components in GL pretreated rice straw at different temperature and TTA charge are shown in Fig. 3. Sugar recovery, lignin removal and silica retention (calculated according to Eqs. (2)–(4) of rice straw after GL pretreatment are given in Table 2:

Silica in pretreated solid ðgÞ  100% Silica in raw material ðgÞ

ð4Þ

Fig. 3 reveals that the recovery of GL pretreated solid declined with the increasing of pretreatment temperature and TTA charge without exception. Compared with temperature, the effect of TTA charge on pretreated solid yield is more significant. It is noted that both glucan and silica keep a high level in GL pretreated solids, while the contents of xylan, arabinan and lignin reduced obviously, especially under severe pretreatment conditions. In traditional soda or kraft pulping process (high pH-value, 13–14), silica in straw materials is easy to be dissolved in spent liquor and it causes a tough problem on extraction, evaporation and combustion of spent liquor for chemical recovery. The pH of pretreatment spent liquor was less than 10 in most cases of this work. As an important benefit of GL pretreatment on straw materials, most silica (68–98% of original silica) could be kept in the pretreated solids. As the pH of sodium acetate buffer was 4.8, all the silica in substrate could be retained in the enzymatic residue after GL pretreatment-enzymatic hydrolysis process. High purity silica nanoparticles could be produced from the hydrolyzed residue by chemical extraction methods or burning directly. This silica product has high surface area and amorphous structure for many industrial applications (Minu et al., 2012; Wattanasiriwech et al., 2010). A great part of polysaccharide could be retained in pretreated solid after rice straw pretreated by green liquor under a mild condition. Table 2 shows that the cellulose (glucan) recovery was higher than 85% even at 16% TTA charge and 140 °C. However, when the temperature was over 140 °C, the degradation of cellulose intensified obviously, especially with a TTA charge of 12–16% as a result of the random alkali degradation and secondary peeling reaction occurred at high temperature and alkalinity (Sjöström, 1993). The recoveries of xylan and arabinan, as the main polysaccharides of hemicellulose in rice straw, were much lower than that of

100 90 80 Content (%)

70 60 50 40 30 20 10

100°C

120°C

140°C

150°C

16

8

12

4

0

16

8

12

0

4

16

8

12

4

0

12

16

8

4

0

12

16

8

4

0

RS

0 160°C

Temperature (°C) and TTA charge (%) of GL pretreatment Glucan

Xylan

Arabinan

KL

ASL

SiO2

Non-SiO2 ash

Extractives

Fig. 3. The content of various components during GL pretreatment at different temperature and TTA charge (RS: rice straw; 0, 4, 8, 12 and 16: percentages of TTA charge in pretreatment; 100, 120, 140, 150 and 160 °C: pretreatment temperature).

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F. Gu et al. / Bioresource Technology 149 (2013) 375–382 Table 2 Effect of GL pretreatment on sugar recovery, lignin removal and silica retention of rice straw (standard deviations are given between brackets). Temp.(°C)

TTA (%)

Sugar recovery (%)

Lignin removal (%)

SiO2 retention (%)

Glucan

Xylan

Arabinan

Total sugar

100

0 4 8 12 16

97.4 96.1 94.9 93.6 91.8

(0.5) (0.4) (0.6) (0.7) (0.9)

99.3 97.2 93.4 90.5 88.7

(0.8) (0.8) (0.7) (0.9) (0.6)

92.9 92.2 92.4 88.9 86.1

(0.5) (0.9) (0.8) (0.8) (0.8)

97.8 96.2 94.3 92.3 90.5

(0.6) (0.7) (0.7) (0.7) (0.7)

6.0 (0.2) 12.0 (0.3) 23.0 (0.3) 36.7 (0.3) 43.6 (0.3)

97.6 98.1 89.1 85.4 78.3

(0.9) (0.7) (0.5) (0.2) (0.4)

120

0 4 8 12 16

95.6 93.4 92.3 91.2 88.7

(0.7) (0.6) (0.8) (0.4) (0.5)

96.0 85.6 75.5 69.0 63.7

(0.8) (0.7) (0.9) (0.4) (0.7)

92.9 90.2 72.8 63.7 57.0

(0.3) (0.5) (0.1) (0.8) (0.6)

95.6 90.7 85.9 82.6 79.0

(0.6) (0.6) (0.6) (0.6) (0.6)

8.2 (0.2) 32.7 (0.3) 55.2 (0.3) 64.9 (0.2) 68.3 (0.3)

95.2 90.3 79.0 73.2 68.9

(0.8) (0.6) (0.4) (0.4) (0.3)

140

0 4 8 12 16

94.0 92.5 90.8 88.2 85.8

(0.4) (0.6) (0.3) (0.5) (0.4)

92.5 82.4 68.4 66.8 63.7

(0.9) (0.7) (0.7) (0.4) (0.5)

89.3 81.6 56.7 54.6 50.5

(0.5) (0.5) (0.9) (0.8) (0.8)

93.3 88.7 81.8 79.6 76.9

(0.6) (0.6) (0.6) (0.7) (0.7)

11.9 39.4 61.0 74.4 76.6

(0.3) (0.2) (0.3) (0.3) (0.4)

95.2 91.8 87.3 74.0 78.4

(0.8) (1.2) (0.3) (0.2) (0.6)

150

0 4 8 12 16

87.2 84.2 81.8 79.7 77.7

(0.3) (0.8) (0.5) (0.6) (0.4)

83.3 75.4 60.9 57.1 55.9

(0.7) (0.9) (0.8) (0.5) (0.8)

82.1 70.5 47.9 41.1 39.8

(0.8) (0.5) (0.4) (0.7) (0.6)

85.7 80.6 73.3 70.4 68.7

(0.7) (0.7) (0.6) (0.6) (0.6)

15.1 48.3 69.3 76.6 79.0

(0.3) (0.3) (0.2) (0.3) (0.3)

97.6 88.6 78.7 74.2 69.9

(0.7) (0.9) (0.1) (0.9) (0.2)

160

0 4 8 12 16

85.2 82.1 79.5 78.5 77.3

(0.5) (0.3) (0.6) (0.8) (0.7)

77.6 66.9 60.7 53.7 54.3

(0.7) (0.4) (0.5) (0.4) (0.4)

50.0 54.7 41.8 38.3 37.8

(0.6) (0.8) (0.7) (0.6) (0.5)

80.9 75.8 71.5 68.4 67.8

(0.6) (0.5) (0.6) (0.5) (0.6)

14.1 54.0 72.4 77.9 78.9

(0.2) (0.2) (0.3) (0.2) (0.3)

97.6 82.3 82.5 75.8 76.8

(0.5) (0.7) (0.5) (0.6) (0.4)

cellulose at the same pretreatment condition, since hemicellulose has lower degree of polymerization, higher branched structures, and more amorphous nature. For example, rice straw pretreated with 4% TTA charge at 140 °C, the recoveries of xylan and arabinan were 82.4% and 81.6%, respectively, and that of glucan was 92.5%. The impacts of pretreatment temperature and TTA charge on the degradation of hemicellulose are much more obvious than that of cellulose. At 150 °C or 160 °C with higher TTA charge (12–16%), about 50% hemicellulose was degraded, in which about 60% of arabinan was dissolved in spent liquor. The total sugar recovery dropped with the increasing of TTA charge due to alkali degradation, especially at high temperature. For example, rice straw pretreated with 12% TTA at 150 °C, only 70.4% of total sugar retained in the recovered solid. The recoveries of glucan, xylan and arabinan were 79.7%, 57.1% and 41.1%, respectively. Lignin has been considered as one of the major barriers for enzymatic hydrolysis, and the delignification in alkali pretreatment usually helps to promote enzymatic hydrolysis (Chang and Holtzapple, 2000). The sugar release in enzymatic hydrolysis is correlated with lignin content, and a strong negative correlation between sugar release and lignin content has been found for alkali pretreated materials (Gu et al., 2012; Jin et al., 2010; Studer et al., 2011). Since the existing of lignin prevents the action of enzyme on polysaccharides by physical hindrance and unproductive enzyme binding (Kumar et al., 2012), delignification is of great importance to obtain ideal sugar yield for alkali pretreatment. The structure of lignin in herbaceous plants is different from that in woody plants. Herbaceous lignin is an amorphous polymer consisting of p-hydroxyphenyl, guaiacyl and syringyl (Lewis and Yamamoto, 1990). It contains some hydroxycinnamic acids (p-coumaric and ferulic acids) in their esterified and etherified form (Lu and Ralph, 2010). Buranov and Mazza (2008) reported that the content of ester-linked p-coumaric acid in rice straw

was 4.9%. This ester linkage is easy to be disrupted even in weak alkali condition. The degradation of lignin is enhanced by the increase of pretreatment temperature and TTA charge. More than three fourths of lignin in rice straw could be removed after the material was pretreated by 12–16% TTA at 140–160 °C with a total sugar recovery of 80%. Compared with other alkali pretreatments (Kim and Han, 2012; Zhu et al., 2005), GL pretreatment has an excellent delignification selectively due to its mild condition. The impacts of pretreatment temperature on lignin removal and sugar degradation are illustrated in Fig. 4. It could be found that GL pretreatment temperature plays an important role on sugar degradation. When the pretreatment temperature was increased from 140 to 150 °C, the enhancement of lignin removal was limited (Fig. 4a) and a great portion of carbohydrate was dissolved (Fig. 4b). It implies that the selectivity of delignification became worse if the pretreatment temperature exceeded 140 °C. In order to achieve a high overall sugar yield, severe sugar degradation in pretreatment ought to be avoided. 3.4. Enzymatic digestibility of GL pretreated rice straw Rice straw pretreated by green liquor under the condition described earlier was hydrolyzed using enzyme cocktail mixed with cellulase, xylanase and b-glucosidase. The enzyme loading based on cellulase activity was 5–40 FPU/g-substrate. The hydrolytic efficiency was evaluated by sugar release and sugar yield, which were calculated by Eqs. (5) and (6), respectively: Sugar release ðmg=g DMÞ ¼

Sugar yield ð%Þ ¼

Sugar in enzymatic hydrolyate ðmgÞ  100% Dry raw material ðgÞ ð5Þ

Sugar in enzymatic hydrolysate ðgÞ  100% Sugar in raw material ðgÞ

ð6Þ

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F. Gu et al. / Bioresource Technology 149 (2013) 375–382

a

b

Fig. 4. Lignin removal (a) and sugar degradation (b) of rice straw in GL pretreatment as functions of pretreatment temperature (0–16%: TTA charge).

Glucan, xylan and total sugar release of GL pretreated rice straw after enzymatic hydrolysis are listed in Table 3. The sugar releases of all GL pretreated samples under different temperatures and TTA charges increased with the enzyme loading from 5 to 40 FPU/g-substrate. Particularly, an obvious increment was obtained when the enzyme loading increased from 5 to 20 FPU/ g-substrate, and the increment leveled off when the enzyme loading was over 20 FPU/g-substrate. The sugar release in enzymatic hydrolysis increased significantly with improving pretreatment temperature from 100 to 140 °C at the same TTA charge, while it tended to become gentle or a little bit down with the continuous rise in temperature. Fig. 5 demonstrates the glucan, xylan and total sugar yield of GL pretreated rice straw with enzyme loading of 40 FPU/g-substrate. At 100 °C, the sugar yield enhanced with the increase of TTA charge from 0% to 16%. When it came to a higher temperature, the reasonable sugar yield appeared around 4% TTA charge, and the trend of sugar yield become flat or decline slightly with the improvement of TTA charge. Raising the temperature to 150 or 160 °C, there is a relative high sugar yield even without alkali addition. However, the subsequent sugar yield has no obvious promotion with the increase of TTA charge at such a high temperature. The result showed that a suitable pretreatment condition is very important to obtain a desirable sugar yield. Although lignin removal and structure disruption during GL pretreatment can promote the enzymatic digestibility of pretreated solids, a severe pretreatment aggravates the degradation of carbohydrates at the same time. It is no doubt that the rise in carbohydrate degradation will reduce the overall sugar yield of enzymatic saccharification. The impact of pretreatment temperature and TTA charge on xylan yield was much more serious than glucan (Fig. 5b). The above result becomes clearer with corroboration from the effects of lignin removal in GL pretreatment on the total sugar yield, as shown in Fig. 6. Delignification is responded to the extent of pretreatment. Lignin removal increasing from 0% to 40% in GL

Table 3 Sugar release of GL pretreated rice straw after enzymatic hydrolysis (standard deviations are given between brackets). Pretreatment

a

Glucan release (mg/g-DM) Enzyme loading, FPU/g-substrate

Total sugara release (mg/g-DM) Enzyme loading, FPU/g-substrate

Xylan release (mg/g-DM) Enzyme loading, FPU/g-substrate

Temp.(°C)

TTA (%)

5

10

20

40

5

100

0 4 8 12 16

58 (4) 83 (2) 106 (2) 120 (1) 145 (3)

71 (2) 104 (3) 142 (2) 157 (1) 177 (4)

77 (4) 119 (5) 161 (3) 183 (2) 209 (5)

80 (2) 125 (3) 174 (3) 196 (3) 219 (5)

16 27 46 62 77

(5) (3) (2) (1) (1)

20 33 53 69 86

(4) (3) (2) (6) (6)

25 40 64 77 96

120

0 4 8 12 16

68 (2) 116 (0) 134 (1) 177 (3) 183 (4)

84 (0) 161 (0) 179 (0) 224 (1) 224 (1)

94 (3) 208 (5) 230 (2) 253 (3) 264 (3)

101 223 255 261 273

(2) (5) (3) (7) (4)

29 72 79 75 78

(3) (4) (2) (6) (4)

44 85 94 82 84

(1) (0) (0) (4) (2)

56 (6) 96 (2) 99 (3) 96 (5) 100 (3)

140

0 4 8 12 16

71 (2) 196 (2) 191 (4) 186 (2) 174 (2)

96 (2) 235 (3) 237 (5) 237 (3) 235 (1)

109 264 277 271 265

(1) (3) (4) (3) (5)

117 283 287 282 276

(4) (6) (5) (8) (9)

45 72 78 82 83

(4) (3) (3) (2) (1)

59 98 95 94 94

(1) (1) (0) (0) (0)

150

0 4 8 12 16

103 143 149 164 149

(3) (2) (4) (1) (1)

146 214 213 207 191

(3) (3) (2) (3) (3)

175 257 255 245 239

(0) (0) (2) (1) (2)

185 268 263 255 248

(2) (5) (5) (4) (5)

62 55 50 54 72

(4) (6) (2) (3) (1)

79 63 70 72 87

160

0 4 8 12 16

129 154 170 172 163

(2) (1) (1) (2) (3)

171 203 212 212 210

(5) (4) (4) (2) (5)

200 260 245 244 233

(2) (3) (2) (1) (3)

215 268 257 251 241

(5) (7) (8) (5) (7)

73 61 57 59 76

(4) (3) (2) (1) (1)

83 70 69 74 86

10

20

40

5

10

20

77 (9) 114 (6) 158 (4) 190 (2) 232 (4)

93 (5) 140 (5) 203 (4) 235 (7) 274 (6)

104 165 234 270 316

(7) (8) (4) (4) (5)

109 174 251 288 330

(5) (6) (7) (8) (9)

61 (2) 99 (9) 100 (4) 104 (8) 102 (7)

100 198 224 262 272

(4) (4) (3) (9) (8)

131 258 285 317 319

(1) (0) (0) (5) (3)

154 316 343 361 377

(8) (7) (5) (8) (6)

166 335 369 378 388

(3) (11) (6) (12) (10)

71 (2) 115 (2) 103 (2) 102 (1) 100 (4)

79 (5) 121 (6) 107 (9) 105 (10) 104 (10)

119 279 279 278 267

(6) (5) (7) (4) (3)

160 346 344 341 339

(3) (4) (5) (3) (1)

185 394 393 384 377

(3) (5) (6) (4) (9)

203 420 407 400 392

(7) (9) (12) (15) (10)

(2) (1) (3) (1) (4)

90 90 85 88 93

(3) (4) (5) (2) (1)

95 97 89 93 94

(3) (6) (6) (5) (6)

169 206 206 226 230

(7) (8) (6) (4) (2)

230 287 291 287 287

(5) (4) (5) (4) (7)

271 357 349 341 343

(3) (4) (7) (3) (3)

287 377 361 356 354

(3) (8) (10) (7) (8)

(3) (2) (2) (1) (3)

93 82 84 89 92

(3) (2) (2) (1) (5)

99 91 88 91 93

(10) (8) (8) (8) (10)

206 223 235 238 247

(6) (4) (3) (4) (4)

260 282 290 294 305

(8) (6) (6) (5) (8)

300 352 338 342 335

(5) (5) (4) (2) (8)

322 369 354 353 345

(12) (10) (12) (10) (11)

(4) (3) (1) (2) (0)

Total sugar is the sum of glucan, xylan, and arabinan. The data of arabinan is not listed in this table.

26 43 68 80 99

(2) (2) (4) (5) (6)

40

F. Gu et al. / Bioresource Technology 149 (2013) 375–382

381

a

b

Fig. 6. Total sugar yield of enzymatic hydrolysis as a function of lignin removal in GL pretreatment.

and hemicellulose were partially removed during the GL pretreatment, dualistic linear regression analysis was performed to express the relationship between enzymatic digestibility of GL pretreated substrate and the removal of lignin (Lig. Rmv.) and hemicellulose (Hemi. Rmv.). The result of statistical analysis is shown as Eq. (7) (the analysis was rejected at a significance level of 0.05 level):

Digestibility ¼ 36:52 þ 0:38 ½Lig: Rmv: þ 0:76 ½Hemi: Rmv:

c

The equation showed that both lignin removal and hemicellulose removal significantly correlate to the enzymatic digestibility, and the significant correlation of hemicellulose removal to the digestibility is more important than that of lignin removal to the digestibility. However, consideration of the relationship between sugar yield and the removal of lignin and hemicellulose (Eq. (8)), it could be find that the removal of lignin in GL process is much more important than that of hemicellulose for a high sugar yield:

Sugar yield ¼ 38:15 þ 0:41 ½Lig: Rmv: þ 0:09 ½Hemi: Rmv:

Fig. 5. Sugar yield of GL pretreated rice straw with enzyme loading of 40 FPU/g-substrate.

pretreatment was beneficial to improve final sugar yield. A higher level of lignin removal is not suggested because the sugar yield reduced as a result of serious sugar degradation under severe pretreatment conditions. This phenomenon revealed that it is very important to control a suitable lignin removal by reasonable pretreatments. Generally, it was believed that lignin removal of 20–65% was sufficient to increase the accessibility of the cellulose to enzymes (Han et al., 1983). Statistical analysis was used to evaluate the relationship between the enzymatic digestibility and GL pretreatment. Enzymatic digestibility is defined as the percentage of sugars in enzymatic hydrolysate to those in the pretreated substrate. Since both lignin

ð7Þ

ð8Þ

The removal of hemicellulose in alkaline pretreatment means the loss of fermentable sugar in the substrate. In order to get the highest final sugar yield, it is very important to keep a balance between the enzymatic digestibility of the substrate and the sugar loss in pretreatment. In this work, rice straw pretreated by green liquor with 4% TTA charge, 20% sulfidity at 140 °C, and hydrolyzed by enzyme cocktail with a cellulase loading of 40 FPU/g-substrate for 48 h, the final sugar yields were 83.9%, 69.6% and 78.0%, respectively for glucan, xylan and total sugar. Compared with woody biomass and other grass material, rice straw shows a strong adaptability to GL pretreatment for obtaining a high sugar yield. Jin et al. (2010) reported that total sugar yield of 79.9% can be achieved after mixed hardwood was pretreated by green liquor (20% TTA, 25% sulfidity, 160 °C) and enzymatically hydrolyzed at the same condition. The sugar yield of GL pretreated mixed hardwood was a bit higher than that of GL pretreated rice straw, it could be attributed to more sugar was retained in the pretreated solid of hardwood (90.6%) than that of rice straw (88.7%). For GL pretreated corn stover (8% TTA, 40% sulfidity, 140 °C), about 70% of the original polysaccharides was converted into fermentable sugars in the subsequent enzymatic hydrolysis (20 FPU/g-pulp, 48 h) (Gu et al., 2012). GL pretreatment has many advantages with powerful competitiveness. Several key properties need to take into consideration for low-cost and advanced pretreatment process (Yang and Wyman,

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2008). With the advantages of highly digestible pretreated solid, no significant sugars degradation, minimum amount of toxic compounds, minimum heat and power requirements, chemicals recovery and non-production of solid-waste residues, GL process meets most of the requirements for an effective pretreatment of lignocellulosic biomass. Meanwhile, the method of GL pretreatment was developed based on the concept of kraft pulping and the proven technique will lower both capital costs and investment risk. 4. Conclusions The cellulose in rice straw was stable during GL pretreatment at a mild condition. After pretreated by 4% TTA at 140 °C, 92.5% of glucan and 82.4% of xylan in the raw material were recovered, while about 40% of lignin was removed and more than 90% of silica was retained in the pretreated solid. After enzymatic hydrolysis with an enzyme loading of 40 FPU/g-substrate, the sugar yield was 83.9% of glucan, 69.6% of xylan and 78.0% of total sugar, respectively. Green liquor process showed an excellent adaptability for the pretreatment of rice straw to improve its enzymatic digestibility. Acknowledgements The authors are grateful for the financial support of the National Natural Science Foundation of China (Grant Nos. 31070512 and 31370571), China Scholarship Council (CSC No. 2011832229) and the Doctorate Fellowship Foundation of Nanjing Forestry University. References Binod, P., Sindhu, R., Singhania, R.R., Vikram, S., Devi, L., Nagalakshmi, S., Kurien, N., Sukumaran, R.K., Pandey, A., 2010. Bioethanol production from rice straw: an overview. Bioresour. Technol. 101, 4767–4774. Buranov, A.U., Mazza, G., 2008. Lignin in straw of herbaceous crops. Ind. Crop. Prod. 28, 237–259. Chang, V.S., Holtzapple, M.T., 2000. Fundamental factors affecting biomass enzymatic reactivity. Appl. Biochem. Biotechnol., 84–86. Chen, L., Xing, L., Han, L., 2009. Renewable energy from agro-residues in China: solid biofuels and biomass briquetting technology. Renew. Sust. Energy Rev. 13, 2689–2695. Chiaramonti, D., Prussi, M., Ferrero, S., Oriani, L., Ottonello, P., Torre, P., Cherchi, F., 2012. Review of pretreatment processes for lignocellulosic ethanol production, and development of an innovative method. Biomass Bioenergy 46, 25–35. Ghose, T., 1987. Measurement of cellulase activities. Pure Appl. Chem. 59, 257–268. Gu, F., Yang, L.F., Jin, Y.C., Han, Q., Chang, H.M., Jameel, H., Phillips, R., 2012. Green liquor pretreatment for improving enzymatic hydrolysis of corn stover. Bioresour. Technol. 124, 299–305. Han, Y.W., Catalano, E.A., Ciegler, A., 1983. Treatments to improve the digestibility of crop residues. In: Soltes, E.J. (Ed.), Wood and Agricultural Residues. Academic Press, pp. 217–238. Hu, F., Ragauskas, A., 2012. Pretreatment and lignocellulosic chemistry. Bioenergy Res. 5, 1043–1066.

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