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Comparison of sodium carbonate pretreatment for enzymatic hydrolysis of wheat straw stem and leaf to produce fermentable sugars
Accepted Manuscript Comparison of sodium carbonate pretreatment for enzymatic hydrolysis of wheat straw stem and leaf to produce fermentable sugars Yongcan Jin, Ting Huang, Wenhui Geng, Linfeng Yang PII: DOI: Reference:
S0960-8524(13)00526-9 http://dx.doi.org/10.1016/j.biortech.2013.03.140 BITE 11600
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
28 December 2012 18 March 2013 20 March 2013
Please cite this article as: Jin, Y., Huang, T., Geng, W., Yang, L., Comparison of sodium carbonate pretreatment for enzymatic hydrolysis of wheat straw stem and leaf to produce fermentable sugars, Bioresource Technology (2013), doi: http://dx.doi.org/10.1016/j.biortech.2013.03.140
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Comparison of sodium carbonate pretreatment for enzymatic hydrolysis of wheat straw stem and leaf to produce fermentable sugars
Yongcan Jin*, Ting Huang, Wenhui Geng, Linfeng Yang
Jiangsu Provincial Key Lab of Pulp and Paper Science and Technology, Nanjing Forestry University, Nanjing 210037, China
[email protected]; [email protected]; [email protected]; [email protected]
* Corresponding author Dr. Yongcan Jin Laboratory of Wood Chemistry Department of Paper Science and Technology Nanjing Forestry University 159 Longpan Rd., Nanjing 210037, China E-mail address: [email protected] Tel.: +86(25)8542 8163 Fax: +86(25)8542 8689
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Abstract
The specific characteristics of biomass structure and chemical composition of straw stem and leaf may result in different behavior of pretreatment and enzymatic hydrolysis. In this work, sodium carbonate (SC) was employed as a pretreatment to improve the enzymatic digestibility of wheat straw. The chemical composition and enzymatic hydrolysis of wheat straw stem and leaf (sheath included) were investigated comparatively. Most of the polysaccharides are kept in the solid fractions after SC pretreatment, while the stem has better delignification selectivity than leaf. The enzymatic hydrolysis efficiency of wheat straw leaf is significantly higher than that of stem. The maximum total sugar yield from SC pretreated leaf was about 16% higher than stem. The results show that sodium carbonate is of great potential to be used as a pretreatment for the production of bioethanol from straw handling waste in a straw pulp mill with a low feedstock cost.
Keywords
Wheat straw; Leaf; Stem; Sodium carbonate pretreatment; Enzymatic hydrolysis
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1. Introduction
The enzymatic hydrolysis process is regarded as the most attractive way to degrade cellulose to glucose. The factors that have been identified to affect the hydrolysis of cellulose include porosity (accessible surface area) of the materials, cellulose fiber crystallinity, and lignin and hemicellulose content (McMillan, 1994; Ragauskas et al., 2006). Cellulose is embedded in lignin, and the presence of lignin is generally considered to be one of the most important limiting factors in the enzymatic cell wall saccharification process (Vanholme et al., 2010; Ding et al., 2012). The concerns about the lignin barrier for enzymatic hydrolysis are typically from three aspects: lignin blocks the accessibility of enzymes, lignin non-productively adsorbs the enzymes, and lignin-carbohydrate complex probably limits the enzymatic hydrolysis (Mooney et al., 1998; Yu et al., 2011). An effective pretreatment is necessary to liberate the cellulose from the lignin seal and its crystalline structure so as to render it accessible for a subsequent hydrolysis step (Gray et al., 2006; Mosier et al., 2005; Kumar et al., 2009). A range of chemical, physical and biological processes to release these sugars have been discussed, yet they all face challenges of cost, technological breakthroughs and infrastructure needs. Chemical pretreatments can remove chemical barriers so that the enzymes can access to cellulose for hydrolysis. Compared to acid pretreatments, alkaline processes have less sugar degradation, furan derivative formation is avoided and many of the caustic salts can be recovered (Gonzalez et al., 1986). Alkaline chemistries have gained renewed interest and are generally more effective in the pretreatment of agricultural residues and herbaceous crops (Chen et al., 2007). Dilute alkaline pretreatment of lignocellulosic materials caused
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swelling, and led to an increase in internal surface area, a decrease in the degree of polymerization and crystallinity, separation of structural linkages between lignin and carbohydrates, and disruption of the lignin structure (Sun and Cheng, 2002).
Green liquor (GL) consists chiefly of sodium carbonate and sodium sulfide (Na2CO3+Na2S). It can be used as pretreatment chemicals for the production of bioethanol from lignocellulosic biomass. The green liquor pretreatment process was developed with the concept of repurposing an old kraft pulp mill for ethanol production, avoiding the closures of some recent pulp mills due to the declining demand in pulp and paper (Jin et al., 2010; Gu et al., 2010). Yang et al. (2012) reported that sodium carbonate was a promising pretreatment for enhancing sugar yield of agricultural residues such as rice straw. During the process of green liquor or sodium carbonate pretreatment, all fermentable sugars are recovered in a single step of enzymatic hydrolysis, without production of fermentation inhibitors. Besides, the inorganic chemicals used are recovered and the dissolved organics including lignin are burned to produce energy, results a minimization of the operating costs.
As one of the world’s most widely grown crops, wheat straw is cultivated in over 115 nations under a wide range of environmental conditions. Over the past 100 years, the yields of wheat have been increased and annual global production of dry wheat in 2008 was estimated to be over 650 Tg (Farid et al., 2010). Wheat straw consists of two major parts, the leaf (leaf and sheath) and the stem. Cavity, parenchyma tissue, vascular bundles, sclerenchyma tissues and epidermis are presented from inner to outer of the stem. The chemical components are significantly different between the outer side and inner side of the
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wheat straw stem, the inner side of the stem is rich in cellulose, and the outer side has more lignin. The contents of cellulose for both sides of the leaf sheath are almost the same (Yu et al., 2007). Besides, the dense layers of the cell wall give additional mechanical strength and higher tensile strength crystalline to the stem (Harper and Lynch, 1981). Since the characteristics of straw materials, such as more parenchyma cells, low lignin and high ash content, are rather different from those of wood, a mild pretreatment could be considered for an acceptable sugar yield in the following enzymatic hydrolysis process.
Based on the conception of GL process, sodium carbonate (SC) is an alternate pretreatment chemical for enhancing sugar yield of straw materials. The chemical can be recovered using a recovery boiler and its use does not require the supplement of sodium sulfate thereby simplifying the overall operation. On the other hand, problems of spent liquor evaporation and combustion in straw soda pulping mills can be effectively avoided since most silica retains in the pretreated solids. In this paper, the effects of sodium carbonate pretreatment at different alkali charge and temperature on the chemical compositions of wheat straw stem and leaf, as well as the sugar yield of enzymatic hydrolysis were comparatively investigated. As there is a large amount of leaf and sheath in the waste residues from straw handling yard, the results will demonstrate the possibility of using these residues as a free raw material for the production of bioethanol.
2. Materials and methods
2.1. Materials
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Wheat straw (Triticum aestivum L.) was obtained from Jiangsu, China. The air dried straw was separated by hand into the stem and leaf (sheath included) carefully and cut to 3–5 cm in length. The samples were collected in sealed plastic bags and stored in a refrigerator at 4 °C. The main component of raw material was analyzed before pretreatment. Three enzymes, cellulase from Trichoderma reesei (NS-50013, 52.3 mg protein/mL, 84 FPU/mL), β-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, USA). All the chemicals were analytical grade and purchased from Nanjing Chemical Reagent Co., Ltd of China and used as received without further purification.
2.2. Sodium carbonate pretreatment
A rotary lab-scale cooking system with an electrically heated oil bath was used for the pretreatment. Ten 1 L-stainless steel bomb reactors with screw cap were contained in the cooking system. The pretreatment was carried out with a batch capacity of 80 g oven dry (od) wheat straw per bomb. The straw samples were directly subjected to pretreatment using sodium carbonate with different total titratable alkali (TTA) charge (6%, 8% and 10%, as Na2O). The ratio of liquor to straw was 6:1 (mL/g). The Na2CO3 concentration of the pretreatment liquors were 17.1, 22.8 and 28.5 g/L, and the corresponding pH-values were 11.19, 11.21 and 11.23, respectively. The materials were first impregnated with the pretreatment liquor at 80 °C for 30 min. Then the temperature was raised with the rate of
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1.5 °C/min to the target temperature (120 °C, 130 °C and 140 °C). The pretreatment was immediately terminated while the designed temperature was reached. At the end of the pretreatment, the solids were collected and washed with hot water to remove residual chemicals and dissolved straw compounds. Pretreated solid yield, defined as the od weight of solid fraction divided by the od weight of original starting material, 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 KRK refiner (Φ 300 mm, 3000 rpm) was used for the defiberization of the pretreated wheat straw samples to produce pulp as substrates. Enzymatic hydrolysis of the substrates was carried out at substrate consistency of 5% (w/w) in sodium acetate buffer (pH 4.8) at 50°C using a shaking incubator at 180 r/min. An enzyme cocktail mixed by NS50013, NS-50010 and NS-50014 was used for enzymatic hydrolysis. The dosage of βglucosidase and xylanase supplementation constituted 30% of the volume of cellulase added (Lee et al., 2010). Enzyme cocktail loading was 10 and 20 FPU/g-cellulose based on cellulase activity. Tetracycline was charged at 40 μg/mL buffer as an antibiotic to inhibit microbial growth during the enzymatic hydrolysis. Enzymatic hydrolysis residue and hydrolysate was separated by centrifugation. Hydrolysates were sampled for monomeric sugar (glucose, xylose and arabinose) analysis.
2.4. Analytical methods
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Cellulase activity in terms of “filter paper unit” (FPU) of cellulase 50013 was determined by the filter paper method using Whatman No. 1 filter paper as a standard substrate (Ghose, 1987). The enzyme protein content was determined using the Bradford method (Smith et al., 1985). Manufacturer specified activity of Novozymes 50014 and 50010 was directly used to calculate loading. The enzymatic hydrolysate was diluted 10 times and monomeric sugars in the hydrolysate were determined using a high performance liquid chromatography (Agilent 1200 Series, Santa Clara, CA) with refractive index detector (RID). A Bio Rad Aminex HPX-87H 20n exclusion column and a Cation-H refill cartridges (Bio-Rad Laboratories, Hercules, CA) were used as analytical column and guard column. The column temperature was 55 °C. A 5 m mol/L H2SO4 solution prepared with degassed super-purified deionized water was used as eluent at a flow rate of 0.6 mL/min. Aliquots (10 μL) were injected after passing through a 0.22 μm nylon syringe filter. 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 glucose, xylose and arabinose contents were corrected to anhydro units, i.e. glucan, xylan and arabinan for the calculation of sugar yield in enzymatic hydrolysate. Sugar yield in enzymatic hydrolysate was reported in weight percent of original glucose, xylose and total sugar in the original staring materials.
The content of benzene-ethanol extractives was determined according to Tappi Standard (T 204 cm-97). Lignin and carbohydrate contents of the raw and pretreated materials were analyzed using the NREL protocol (Sluiter et al., 2008). The Klason lignin content was
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taken as the ash free residue after acid hydrolysis. The ash content was determined by combusting the samples in a furnace at 575 °C. The hydrolysate from this determination was retained for analysis of sugars and acid-soluble lignin (AS lignin). Sugars were determined by HPLC 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. To meet the requirement of column pH, 40 μL 50% (w/w) NaOH solution was injected per mL of hydrolysate sample prior to sugar analysis. Acid-soluble lignin was measured by absorbance at 205 nm in a 765 UV-VIS spectrometer. Each data point was the average of duplicate experiments.
3. Results and discussion
3.1. Chemical composition of wheat straw
The composition of wheat straw, wheat straw stem and wheat straw leaf (leaf and sheath included) used in this study is given in Table 1.
The composition of wheat straw, like other lignocellulosic biomass, is a complex mixture of cellulose, hemicellulose and lignin as three major components, and a small amount of extractives and ash. The benzene-ethanol extractives and ash in leaf were 6.5 and 11.3%, respectively, and those in stem were 4.6 and 7.6%, respectively. The higher content of extractives and ash in leaf is due to the higher content of epidermal cell, which is mainly composed by suberized cells and silica cells. Glucan and xylan are the two main
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polysaccharides in wheat straw. The xylan content of stem was almost as the same as that of leaf, however, the glucan in stem was obviously higher than that in leaf. It means that compared with leaf, wheat straw stem contains more cellulose. Arabinan in stem was lower than that in leaf. The result indicates that, for wheat straw, the xylan structure of stem is different from that of leaf in some degree. The total lignin of stem was 23.0%, which was 4.9% higher than that of leaf (18.1%). Compared with stem, leaf had a higher content of Klason lignin and a lower content of acid-soluble lignin. The ratio of Klason lignin and acid-soluble lignin in stem was 9.2:1, while it was 5.7:1 in leaf. The sum of polysaccharides and lignin in stem was 86.0%, which was 10.1% higher than that in leaf.
3.2. Effect of sodium carbonate pretreatment on chemical composition of wheat straw
In this work, wheat straw, stem and leaf were directly subjected to pretreatment using sodium carbonate charge varied from 6% to 10% at 120 °C, 130 °C and 140 °C on the basis of bone dry materials. The effect of pretreatment temperature and TTA charge on the SC pretreated solid yield is shown in Fig. 1.
The pretreated solid yield of the different part of wheat straw under described conditions was approximately in the range of 60–90%. Random alkaline hydrolysis and secondary peeling reactions (Sjöström, 1993) did not occur in the sodium carbonate pretreatment as a result of the much lower pH-value of pretreatment liquor in comparison with that of the conventional kraft and soda cooking liquor.
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The pretreated solid yield decreased with the increment of sodium carbonate charge and pretreatment temperature. As the alkalinity of sodium carbonate is weak, the increasing of pretreatment TTA charge from 6% to 10% at 120 °C only resulted in a small decrease of pretreated solid yield (ranged from 85.8% to 81.7%). However, when the pretreatment temperature was raised from 120 °C to 140 °C at 6% TTA charge, there was an obvious decrease of pretreated solid yield (ranged from 85.8% to 72.6%). It reveals that compared to the pretreatment temperature, the effect of TTA charge is not significant on the solid yields of wheat straw.
The SC pretreated solid yield of leaf was much lower than that of stem at the same temperature and TTA charge. It is mainly caused by the higher contents of benzene-ethanol extractives and ash in leaf in comparison with those in stem, as the benzene-ethanol extractives and the inorganic substances were easily dissolved at the pretreatment conditions. The yield of leaf pretreated with 10% TTA charge at 140 °C was 62.9%, which was the lowest among all the samples in this work. Harsh conditions during pretreatment resulted in a partial cellulose and hemicellulose degradation that could affect the following enzymatic hydrolysis (Lynd, 1996).
The effect of sodium carbonate pretreatment on the delignification of wheat straw was quantified by the reduction of lignin content in pretreated solids as a function of pretreatment temperature and TTA charge. As shown in Fig. 2, a good linear relationship can be observed for the solids pretreated by 6% to 10% TTA charge at 120 °C to 140 °C. At 140 °C, the lignin removal ranged from 35.1% at 6% TTA to a maximum of 44.7% at
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10% TTA of the SC pretreated wheat straw, similar reductions in solid yields were attained irrespective of the leaf and stem. There was no obvious difference of the delignification selectivity among the wheat straw, stem and leaf samples pretreated with different TTA charge at 120 °C and 130 °C. The delignification selectivity of SC pretreated leaf became worse with the increase of pretreatment temperature. The pretreated solid yield dropped significantly at a same lignin removal level when the pretreatment temperature reached to 140 °C. Compared to the SC pretreated leaf in the same condition, the stem had a better delignification selectivity. For example, the pretreated solid yield of stem was 72.0% with a lignin removal of 39.1% when pretreated by 8% TTA at 140 °C; However, that of leaf was only 67.5% with a similar lignin content of 37.8% when pretreated by 6% TTA at 140 °C. It means that since straw lignin is easy to be removed even at a relatively low temperature, High pretreatment temperature does not help to improve delignification, but lowers the pretreated solid yield.
The percentage of each chemical component through SC pretreatment processes (TTA charge 6–10%, pretreatment temperature 120–140 °C) based on the original weight of different materials is presented in Fig. 3. It reveals that the losses of polysaccharides and lignin increase with the increment of TTA charge and pretreatment temperature. After the wheat straw was pretreated by 10% TTA charge at 140 °C, the degradation degree of glucan was approximately 10%, while xylan was over 28%. As briefly discussed previously, cellulose is more stable than hemicellulose under the SC pretreatment condition. Hemicellulose is relatively labile and dissolved in SC pretreatment at 140 °C.
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Alkaline pretreatment partially solubilized the hemicellulose fraction and gave birth to a material enriched in cellulose (Silverstein et al., 2007; Varga et al., 2003).
Lignin is easily to be degraded under alkaline conditions. For example, the lignin removal of the SC pretreated wheat straw was 23.2% at 120 °C with 6% TTA charge, while the degree of polysaccharides degradation was only 1.2%. Meanwhile, the degrees of lignin and polysaccharides degradation at 140 °C with 10% TTA were 44.7 and 17.6%, respectively. It indicates that the degradation of lignin is higher than that of cellulose and hemicellulose during SC pretreatment, especially under a mild pretreatment condition. However, pretreatment under severe condition such as high temperature and high chemical charge resulted in an increasing degradation of polysaccharides. A high retention of sugar with a certain delignification is the principle point to an effective pretreatment for bioethanol production (Nigam, 2001; Tabka et al., 2006). The ash content of SC pretreated wheat straw was 4.7% at 120 °C with 6% TTA charge, which was only 1.3% higher than that at 140 °C with 10% TTA charge.
The comparison of main chemical components between SC pretreated leaf and stem under different pretreatment conditions are shown in Figs. 3(b) and 3(c). The degradation of carbohydrate was little for both stem and leaf when the samples were pretreated at 120 °C, and it became severe when the pretreatment temperature increased to 140 °C. More ash was retained in pretreated leaf solid in comparison with stem due to the high ash content in the original material. Lignin in leaf is easy to be removed even under a mild pretreatment condition. When the TTA charge ranges from 6% to 10% and the temperature ranged from
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120 °C to 140 °C, the highest lignin removal of the SC pretreated leaf and stem were 58.1% and 44.7%, respectively. As lignin acts as a physical barrier to protect cellulose against microbial or chemical degradation (Mooney et al., 1998; Yu et al., 2011; Ding et al., 2012), the higher lignin content of the SC pretreated stem sample may influence the efficiency of the following enzymatic hydrolysis.
3.3. Effect of sodium carbonate pretreatment on the enzymatic hydrolysis of wheat straw
The SC pretreated solids were refined to produce pulp as substrate for enzymatic hydrolysis. The pulps were hydrolyzed by the enzyme mixture of cellulase, xylanase and β-glucosidase to evaluate the improvement of sugar yield. The xylanase used in the experiment was a little excessive. Supplementing the enzyme mixture with additional xylanase activity will improve saccharification of mildly treated samples containing higher amounts of xylan (Bura et al., 2009). The effects of sodium carbonate pretreatment temperature and TTA charge on sugar yield in enzymatic hydrolysate of SC pretreated wheat straw, stem and leaf are given in Tables 2 to 4.
The enzyme loadings of 10 FPU/g-cellulose and 20 FPU/g-cellulose were selected to evaluate the effect of sodium carbonate pretreatment on enzymatic hydrolysis. Total sugar in the tables is the sum of glucan, xylan and arabinan. The arabinan content is not listed since it is much lower than glucan and xylan. It is evident that the effect of enzyme dosage was more pronounced at 20 FPU/g-cellulose than at 10 FPU/g-cellulose. An obvious
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increment of sugar yield could be observed for all SC pretreated samples when the enzyme loading was raised from 10 FPU/g-cellulose to 20 FPU/g-cellulose.
Table 2 shows that the total sugar yield of SC pretreated wheat straw pulp at 120 °C with 6% TTA charge was 57.6%, under an enzyme loading of 20 FPU/g-cellulose. The total sugar yield could be improved to 61.7% by increasing the TTA charge from 6% to 8%. There was no much difference between 8% and 10% TTA charge on the total sugar yield. Same situation was observed for the pulps pretreated at 130 °C and 140 °C. So it is clear that 8% TTA charge was a critical dosage for the pretreatment of wheat straw. The total sugar yield from wheat straw by SC pretreatment (8% TTA, 130 °C) and enzymatic hydrolysis (50 °C, pH 4.8, 48 h) using 20 FPU/g-cellulose was 64.8% (Table 2). It was the highest one among all pretreated wheat straw samples.
However, continuously increasing TTA charge from 6% to 10% of SC pretreated stem further improved the bioconversion of cellulose and hemicellulose to glucose and xylose (Table 3). In the condition of pretreatment temperature 130 °C and TTA charge 10%, the total sugar yield of the SC pretreated stem was 58.7%, which was the highest among all the pretreated stem samples. The glucan and xylan yield in hydrolysate were 56.2% and 62.1%, respectively in this condition. It could be calculated that about 39.1% of the cellulose and 23.4% of the hemicellulose were retained in the residue after enzymatic hydrolysis. The further pretreatment development is recommended to focus on the increase of cellulose accessibility, such as improving the TTA charge of SC pretreatment at 130 °C on wheat straw stem and the higher total sugar yield could be expected.
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In contrast to the enzymatic hydrolysis efficiency of the stem, SC pretreatment on the leaf resulted in a larger increment in the total sugar yield of enzymatic hydrolysis (Tables 3 and 4). For example, at 130 °C with 10% TTA charge, the total sugar yield in hydrolysate of the SC pretreated leaf pulp was 73.1%, which was 14.4% higher than that of the pretreated stem pulp. The lignin removal of the SC pretreated leaf and stem under this condition were 50.0% and 31.5%, respectively. Lignin removal has been reported to improve cellulose accessibility effectively by creating pores and breaking the lignin-carbohydrate complex (Yang and Wyman, 2004). An increase in the surface area of the cellulose accessible to the cellulase would have a considerable effect on hydrolysis, as the enzyme requires contact with the substrate for hydrolysis to occur (Chandra et al., 2007). The enzymatic hydrolysis of leaf pulp pretreated with sodium carbonate (120 °C, 8% TTA charge) released 74.6% of total sugar, which was the highest among all the samples (Table 4). This research elucidates that straw leaf subjected by SC pretreatment can be easily converted to monomeric sugars during the enzymatic saccharification. The significant difference between the total sugar yield of leaf and stem indicates that the enzymatic hydrolysis efficiency of the wheat straw leaf is much better than that of stem.
The alkalinity of sodium carbonate is not strong enough to serve as an ideal pretreatment for wheat straw. In this work, the highest total sugar yields of SC pretreated wheat straw were 62.1%, 67.9% and 64.8%, respectively. It is lower than the sugar yield of wheat straw pretreated by sodium hydroxide or lime (Saha and Cotta, 2007; McIntosh and Vancov, 2011). Pedersen et al. (2010) found that the solid fraction was more susceptible to
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enzymatic attack when wheat straw was pretreated by sodium hydroxide at pH 13. After hydrolyzed by a standard blend of Celluclast 1.5L and Novozyme 188, the glucose, xylose and total sugar yield obtained from enzymatic hydrolysis of the solid and liquid fraction were 76%, 92% and 80.1%, respectively. However, the total sugar yield became to 55.7% if the dissolved cellulose and hemicellulose in liquid fraction was not recovered. The total sugar yield of 64.8% is also lower than SC pretreated rice straw. Yang et al. (2012) reported that the sugar yield from rice straw by SC pretreatment (TTA 8%, 140 °C) and enzymatic hydrolysis (50 °C, pH 4.8, 48 h) using a cocktail of the same three enzymes at a loading of 20 FPU/g-cellulose was 71.7%. The higher portion of leaf and sheath in rice straw might be the key reason. As discussed earlier, SC pretreated leaf and sheath of wheat straw exhibits better efficiency of enzymatic saccharification than stem.
Soda pulping is regarded as a proven process to produce pulp using straw fiber as raw materials. Leaf and sheath should be removed as much as possible in straw handling yard to improve the quality of straw pulp. The results of this work show that straw handling rejects in a soda pulp mill is of great potential to be used as a cheap feedstock for the production of bioethanol using sodium carbonate as a pretreatment. A co-location bioethanol line using straw handling waste as raw materials could be considered in an operating soda pulp mill, as the pretreatment chemical, sodium carbonate can be recycled in the proven alkali recovery system. It will effectively cut the operation cost in bioethanol production.
3.4. Effect of lignin removal on enzymatic hydrolysis
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The removal of lignin by SC pretreatment in this study may result in enhanced enzymatic hydrolysis by reducing the cellulase-lignin binding and increasing the accessibility of cellulose to the enzyme. It is reported that removal of 20–65% of the lignin in a sample is sufficient to increase the accessibility of the cellulose to enzymes (Ko et al., 2009). Fig. 4 illustrates the effect of lignin removal on total sugar yield of enzymatic hydrolysis at enzyme loadings of 10 FPU/g-cellulose and 20 FPU/g-cellulose. The total enzymatic hydrolysis sugar yield was increased with the lignin removal when the lignin removal was less than 30%. For most SC pretreated samples, the total sugar yield reached the highest when the lignin removal ranged from 30% to 40%. When the lignin removal of SC pretreated solids was more than 40%, enzymatic hydrolysis nearly leveled off. This is mainly due to the cleavage of aryl ether linkage of lignin accelerated by alkali during SC process, more lignin is fragmented and dissolved, and more surface area of carbohydrates is exposed. However, further delignification along with the decrement of pretreated solid yield influences the overall sugar yield of enzymatic hydrolysis of SC pretreated samples.
When comparing the total sugar yield to the same lignin basis, the SC pretreated leaf was more effective in enzymatic hydrolysis than stem. As shown in Fig. 4, when the lignin removal of SC pretreated stem pulp was 26.4% (130 °C, 8% TTA charge), the total sugar yield was only 56.3% after hydrolyzed with 20 FPU/g-cellulose. However, the total sugar yield of SC pretreated leaf pulp reached to 69.5% at a similar lignin content of 27.1% (120 °C, 6% TTA charge). In addition, there was a sharp increase of the lignin removal accompany with the decrease of the total sugar yield when the temperature reached to 140 °C. The highest total sugar yield of SC pretreated leaf solids (8% Na2CO3, 120 °C) was
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74.5%. It was about 16% higher than the highest one of SC pretreated stem pulp (10% Na2CO3, 130 °C).
4. Conclusions
The chemical composition of wheat straw leaf is rather different from that of stem. Most of the polysaccharides are kept in the pretreated solids, and lignin in leaf is easy to be removed during SC pretreatment. The epretreated wheat straw leaf exhibits higher enzymatic hydrolysis efficiency in comparison with stem. The highest yield of total sugar from wheat straw leaf by SC pretreatment (8% TTA, 120 °C) and enzymatic hydrolysis (20 FPU/g-cellulose) was 74.5%, which was 16% higher than that from stem. The result provides a practical pretreatment pathway to produce bioethanol using the waste from straw handling yard.
Acknowledgements
The authors are grateful for the financial support of the National Basic Research Program of China (973 Program, Grant No. 2010CB732205), the National Natural Science Foundation of China (Grant No. 31070512), and the Priority Academic Program Development of Jiangsu Higher Education Institutions.
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17. McMillan, J.D., 1994. Pretreatment of lignocellulosic biomass. ACS Symp. Ser. 566, 292–324. 18. Mooney, C.A., Mansfield, S.D., Touhy, M.G., Saddler, J.N., 1998. The effect of initial pore volume and lignin content on the enzymatic hydrolysis of softwoods. Bioresour. Technol. 64, 113–119. 19. Mosier, N., Wyman, C., Dale, B., Elander, R., Lee, Y.Y., Holtzapple, M., Ladisch, M., 2005. Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresour. Technol. 96, 673–686. 20. Nigam, J.N., 2001. Ethanol production from wheat straw hemicellulose hydrolysate by Pichia stipitis. J. Biotechnol. 87, 17–27. 21. Pedersen, M., Viksø-Nielsen, A., Meyer A.S., 2010. Monosaccharide yields and lignin removal from wheat straw in response to catalyst type and pH during mild thermal pretreatment. Process Biochem. 45, 1181–1186. 22. Ragauskas, A.J., Williams, C.K., Davison, B.H., Britovsek, G., Cainey, J., Eckert, C.A., Frederick, W.J., Hallett, J.P., Leak, D.J., Liotta, C.L., Mielenz, J.R., Murphy, R., Templer, R., Tschaplinski, T., 2006. The path forward for biofuels and biomaterials. Science 311, 484–489. 23. Saha, B.C., Cotta, M.A., 2007. Enzymatic hydrolysis and fermentation of lime pretreated wheat straw to ethanol. J. Chem. Technol. Biotechnol. 82, 913–919. 24. Silverstein, R.A., Chen, Y., Sharma-Shivappa, R.R., Boyette, M.D., Osborne, J.A., 2007. Comparison of chemical pretreatment methods for improving saccharification of cotton stalks. Bioresour. Technol. 98, 3000–3011.
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25. Sjöström, E., 1993. Wood Chemistry: Fundamentals and Applications. Academic Press, New York, pp. 150–155. 26. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Crocker, D., 2008. Determination of structural carbohydrates and lignin in biomass, Laboratory Analytical Procedure, NREL Report No. TP-510-42618. 27. Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., Klenk, D.C., 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76–85. 28. Sun, Y., Cheng, J., 2002. Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresour. Technol. 83, 1–11. 29. Tabka, M.G., Herpoel-Gimbert, I., Monod, F., Asther, M., Sigoillot, J.C., 2006. Enzymatic saccharification of wheat straw for bioethanol production by a combined cellulase xylanase and feruloyl esterase treatment. Enzyme Microb. Technol. 39, 897– 902. 30. Vanholme, R., Demedts, B., Morreel, K., Ralph, J., Boerjan, W., 2010. Lignin biosynthesis and structure. Plant Physiol. 153, 895–905. 31. Varga, E., Schmidt, A., Reczey, K., Thomsen, A., 2003. Pretreatment of corn stover using wet oxidation to enhance enzymatic digestibility. Appl. Biochem. Biotechnol. 104, 37–50. 32. Yang, B., Wyman, C.E., 2004. Effect of xylan and lignin removal by batch and flow through pretreatment on the enzymatic digestibility of corn stover cellulose. Biotechnol. Bioen. 86, 88–98.
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33. Yang, L., Cao, J., Jin, Y., Chang, H.-m., Jameel, H., Phillips, R., Li, Z., 2012. Effects of sodium carbonate pretreatment on the chemical compositions and enzymatic saccharification of rice straw. Bioresour. Technol. 124, 283–291. 34. Yu, H., Liu, R.G., Qiu, L.M., Huang, Y., 2007. Composition of the cell wall in the stem and leaf sheath of wheat straw. J. Appl. Polym. Sci. 104, 1236–1240. 35. Yu, Z.Y., Jameel, H., Chang, H.-m., Park, S., 2011. The effect of delignification of forest biomass on enzymatic hydrolysis. Bioresour. Technol. 102, 9083–9089.
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Figure captions
Fig. 1. Effects of TTA charge and temperature on SC pretreated solid yield. Samples obtained at different pretreatment temperature from wheat straw, stem and leaf are expressed as W-T, S-T and L-T, in which T is pretreatment temperature. Fig. 2. Lignin removal as a function of sodium carbonate pretreated solid yield. Samples obtained at different pretreatment temperature from wheat straw, stem and leaf are expressed as W-T, S-T and L-T, in which T is pretreatment temperature. The three data points on each line are corresponding to the different TTA charge of pretreatment: 6%, 8% and 10% (from left to right). Fig. 3. Retention of various components in sodium carbonate pretreated wheat straw (a), stem (b) and leaf (c). Fig. 4. Effect of lignin removal on total sugar yield of enzymatic hydrolysis at cellulase loading of 10 FPU/g-cellulose (a) and 20 FPU/g-cellulose (b). Samples obtained at different pretreatment temperature from wheat straw, stem and leaf are expressed as W-T, S-T and L-T, in which T is pretreatment temperature. The three data points on each line are corresponding to the different TTA charge of pretreatment: 6%, 8% and 10% (from left to right).
25
Fig. 1.
100
80
70
60
50 6 8 TTA (%) 10 L-140
L-130
L-120
W-140
W-130
Samples
26
W-120
S-140
S-130
S-120
Pretreated solid yield (%)
90
Fig. 2. 70 L-120 L-130 L-140
Lignin removal (%)
60 50
W-120 W-130 W-140
S-120 S-130 S-140
40 30 20 10 0 60
65
70
75
80
85
Pretreated solid yield (%)
27
90
95
Fig. 3. 100 90 80 Ccontent (%)
70 60 50 40 30 20 10 0 Raw
6%
8%
10%
120˚C
6%
8%
10%
6%
130˚C Wheat straw
8%
10%
140˚C
(a) 100 90 80 Content (%)
70 60 50 40 30 20 10 0 Raw
6%
8%
10%
6%
120˚C
8% 130˚C
Stem
(b)
28
10%
6%
8% 140˚C
10%
100 90 80
Content (%)
70 60 50 40 30 20 10 0 Raw
6%
8%
10%
6%
120˚C
Glucan AS lignin
8%
10%
130˚C Leaf Arabinan Extractive
Xylan Ash
(c)
29
6%
8%
10%
140˚C
Klason lignin
Fig. 4. 80
Total sugar yield (%)
70 60 50 40 L-120 W-120 S-120
30
L-130 W-130 S-130
L-140 W-140 S-140
20 0
20
40
60
80
Lignin removal in pretreatment (%)
(a) 80
Total sugar yield (%)
70 60 50 40 L-120 W-120 S-120
30
L-130 W-130 S-130
L-140 W-140 S-140
20 0
20
40
60
Lignin removal in pretreatment (%)
(b)
30
80
Table 1 The main chemical components of wheat straw samples (w/w, %). Standard deviations are given between brackets. Benzeneethanol Extractives Wheat straw 5.0 (0.4) Stem 4.6 (0.2) Leaf 6.5 (0.4) Samples
Lignin AcidKlason soluble 19.0 (0.3) 2.5 (0.0) 20.8 (0.1) 2.2 (0.0) 15.4 (0.0) 2.7 (0.0)
Sugar Total
Glucan
Ash Xylan
Arabinan Total
21.5 (0.3) 37.9 (0.5) 19.9 (0.2) 2.9 (0.2) 60.7 (0.5) 8.0 (0.6) 23.0 (0.1) 40.4 (0.1) 20.1 (0.2) 2.5 (0.3) 63.0 (0.6) 7.6 (0.2) 18.1 (0.0) 34.9 (0.2) 19.8 (0.2) 3.0 (0.2) 57.7 (0.1) 11.3 (0.7)
31
Table 2 Effects of pretreatment temperature and TTA charge on sugar yield in enzymatic hydrolysate of SC pretreated wheat straw. Standard deviations are given between brackets. Cellulase loading 10 FPU/g-cellulose Cellulase loading 20 FPU/g-cellulose
Temp. (°C)
TTA charge in pretreatment (%)
Glucan (%) Xylan (%) Total a (%) Glucan (%) Xylan (%) Total (%)
120
6
46.7 (0.1)
45.1 (0.3)
47.5 (0.1)
57.9 (0.3)
55.5 (1.6)
57.6 (0.6)
8
47.7 (0.1)
47.8 (3.2)
51.7 (2.1)
59.2 (0.2)
61.9 (0.1)
61.7 (0.7)
10
44.5 (0.8)
54.9 (1.0)
50.1 (0.6)
58.9 (0.2)
62.3 (0.5)
62.3 (0.3)
6
46.3 (1.7)
48.8 (1.9)
47.3 (1.7)
55.7 (2.9)
56.4 (1.6)
55.9 (2.3)
8
47.1 (0.2)
56.7 (0.7)
51.2 (0.9)
62.1 (0.7)
67.9 (2.0)
64.8 (0.1)
10
46.1 (1.4)
56.7 (2.1)
51.1 (1.5)
61.2 (0.3)
65.9 (0.7)
63.6 (0.1)
6
42.5 (0.6)
45.0 (2.0)
45.7 (1.3)
54.9 (1.1)
52.1 (2.3)
56.6 (1.3)
8
43.5 (0.6)
47.9 (0.5)
47.1 (0.9)
57.8 (1.6)
54.7 (1.9)
59.4 (1.9)
10
42.8 (0.1)
45.3 (1.0)
46.0 (0.2)
56.8 (1.1)
52.3 (0.4)
57.6 (0.8)
130
140
a
Total sugar is the sum of glucan, xylan and arabinan. Data of arabinan are not listed due to
the low content.
32
Table 3 Effects of pretreatment temperature and TTA charge on sugar yield in enzymatic hydrolysate of SC pretreated wheat straw stem. Standard deviations are given between brackets. Cellulase loading 10 FPU/g-cellulose Cellulase loading 20 FPU/g-cellulose
Temp. (°C)
TTA charge in pretreatment (%)
Glucan (%) Xylan (%) Total (%)
Glucan (%) Xylan (%) Total (%)
120
6
38.1 (1.6)
41.8 (1.2)
39.2 (0.6)
46.4 (0.5)
48.1 (0.4)
47.8 (0.8)
8
33.8 (1.1)
51.6 (1.4)
40.6 (0.1)
48.8 (0.5)
59.6 (1.2)
53.2 (0.8)
10
33.7 (0.6)
53.6 (1.2)
41.0 (0.1)
51.2 (0.0)
62.0 (0.0)
55.7 (0.0)
6
35.3 (0.5)
49.4 (0.4)
40.5 (0.5)
53.8 (1.1)
60.2 (0.0)
56.0 (0.6)
8
36.4 (0.3)
50.9 (0.1)
41.9 (0.2)
54.3 (0.7)
59.7 (0.1)
56.3 (0.5)
10
36.6 (0.6)
51.4 (1.8)
42.1 (0.9)
56.2 (1.0)
62.1 (2.1)
58.7 (1.0)
6
35.5 (0.5)
44.7 (0.8)
38.7 (0.1)
52.2 (0.6)
51.2 (0.1)
52.1 (0.4)
8
35.1 (0.1)
47.4 (0.3)
39.7 (0.0)
54.1 (0.7)
55.8 (1.1)
54.7 (0.7)
10
34.5 (0.6)
46.3 (1.3)
38.7 (0.1)
52.5 (0.9)
56.8 (0.2)
54.0 (0.2)
130
140
33
Table 4 Effects of pretreatment temperature and TTA charge on sugar yield in enzymatic hydrolysate of SC pretreated wheat straw leaf. Standard deviations are given between brackets. Cellulase loading 10 FPU/g-cellulose Cellulase loading 20 FPU/g-cellulose
Temp. (°C)
TTA charge in pretreatment (%)
Glucan (%) Xylan (%) Total (%)
Glucan (%) Xylan (%) Total (%)
120
6
65.7 (0.8)
51.8 (0.6)
60.4 (0.5)
75.7 (0.4)
60.0 (0.3)
69.6 (0.5)
8
64.6 (0.4)
59.5 (0.3)
62.8 (0.3)
78.7 (0.1)
67.9 (0.6)
74.6 (0.3)
10
63.3 (0.0)
56.8 (0.2)
61.0 (0.0)
79.5 (0.4)
68.2 (3.0)
74.4 (1.2)
6
65.4 (0.8)
53.3 (0.2)
60.5 (0.4)
79.0 (0.5)
63.1 (1.3)
72.3 (0.7)
8
65.3 (0.6)
57.1 (0.9)
61.7 (0.8)
79.9 (0.4)
65.0 (0.7)
73.8 (0.3)
10
64.6 (0.9)
55.1 (0.9)
60.1 (0.9)
79.3 (1.1)
64.0 (0.2)
73.1 (0.8)
6
61.9 (1.2)
46.3 (2.1)
55.3 (1.4)
70.5 (1.7)
52.0 (1.6)
65.0 (1.5)
8
61.0 (0.6)
47.9 (0.2)
58.7 (0.9)
70.7 (1.7)
53.3 (0.3)
67.9 (1.0)
10
59.8 (0.7)
46.8 (0.9)
56.8 (0.6)
70.8 (1.0)
54.7 (2.9)
67.6 (1.4)
130
140
34
The highlights of the paper are: • Sodium carbonate (SC) was used as a mild alkaline pretreatment on wheat straw; • Chemical composition of SC pretreated wheat straw stem and leaf was compared; • Lignin in leaf was easier to be removed than that in stem; • Enzymatic hydrolysis of SC pretreated wheat straw stem and leaf was compared; • Sugar yield of pretreated wheat straw leaf is much higher than that of stem.
35
S0960-8524(13)00526-9 http://dx.doi.org/10.1016/j.biortech.2013.03.140 BITE 11600
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
28 December 2012 18 March 2013 20 March 2013
Please cite this article as: Jin, Y., Huang, T., Geng, W., Yang, L., Comparison of sodium carbonate pretreatment for enzymatic hydrolysis of wheat straw stem and leaf to produce fermentable sugars, Bioresource Technology (2013), doi: http://dx.doi.org/10.1016/j.biortech.2013.03.140
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Comparison of sodium carbonate pretreatment for enzymatic hydrolysis of wheat straw stem and leaf to produce fermentable sugars
Yongcan Jin*, Ting Huang, Wenhui Geng, Linfeng Yang
Jiangsu Provincial Key Lab of Pulp and Paper Science and Technology, Nanjing Forestry University, Nanjing 210037, China
[email protected]; [email protected]; [email protected]; [email protected]
* Corresponding author Dr. Yongcan Jin Laboratory of Wood Chemistry Department of Paper Science and Technology Nanjing Forestry University 159 Longpan Rd., Nanjing 210037, China E-mail address: [email protected] Tel.: +86(25)8542 8163 Fax: +86(25)8542 8689
1
Abstract
The specific characteristics of biomass structure and chemical composition of straw stem and leaf may result in different behavior of pretreatment and enzymatic hydrolysis. In this work, sodium carbonate (SC) was employed as a pretreatment to improve the enzymatic digestibility of wheat straw. The chemical composition and enzymatic hydrolysis of wheat straw stem and leaf (sheath included) were investigated comparatively. Most of the polysaccharides are kept in the solid fractions after SC pretreatment, while the stem has better delignification selectivity than leaf. The enzymatic hydrolysis efficiency of wheat straw leaf is significantly higher than that of stem. The maximum total sugar yield from SC pretreated leaf was about 16% higher than stem. The results show that sodium carbonate is of great potential to be used as a pretreatment for the production of bioethanol from straw handling waste in a straw pulp mill with a low feedstock cost.
Keywords
Wheat straw; Leaf; Stem; Sodium carbonate pretreatment; Enzymatic hydrolysis
2
1. Introduction
The enzymatic hydrolysis process is regarded as the most attractive way to degrade cellulose to glucose. The factors that have been identified to affect the hydrolysis of cellulose include porosity (accessible surface area) of the materials, cellulose fiber crystallinity, and lignin and hemicellulose content (McMillan, 1994; Ragauskas et al., 2006). Cellulose is embedded in lignin, and the presence of lignin is generally considered to be one of the most important limiting factors in the enzymatic cell wall saccharification process (Vanholme et al., 2010; Ding et al., 2012). The concerns about the lignin barrier for enzymatic hydrolysis are typically from three aspects: lignin blocks the accessibility of enzymes, lignin non-productively adsorbs the enzymes, and lignin-carbohydrate complex probably limits the enzymatic hydrolysis (Mooney et al., 1998; Yu et al., 2011). An effective pretreatment is necessary to liberate the cellulose from the lignin seal and its crystalline structure so as to render it accessible for a subsequent hydrolysis step (Gray et al., 2006; Mosier et al., 2005; Kumar et al., 2009). A range of chemical, physical and biological processes to release these sugars have been discussed, yet they all face challenges of cost, technological breakthroughs and infrastructure needs. Chemical pretreatments can remove chemical barriers so that the enzymes can access to cellulose for hydrolysis. Compared to acid pretreatments, alkaline processes have less sugar degradation, furan derivative formation is avoided and many of the caustic salts can be recovered (Gonzalez et al., 1986). Alkaline chemistries have gained renewed interest and are generally more effective in the pretreatment of agricultural residues and herbaceous crops (Chen et al., 2007). Dilute alkaline pretreatment of lignocellulosic materials caused
3
swelling, and led to an increase in internal surface area, a decrease in the degree of polymerization and crystallinity, separation of structural linkages between lignin and carbohydrates, and disruption of the lignin structure (Sun and Cheng, 2002).
Green liquor (GL) consists chiefly of sodium carbonate and sodium sulfide (Na2CO3+Na2S). It can be used as pretreatment chemicals for the production of bioethanol from lignocellulosic biomass. The green liquor pretreatment process was developed with the concept of repurposing an old kraft pulp mill for ethanol production, avoiding the closures of some recent pulp mills due to the declining demand in pulp and paper (Jin et al., 2010; Gu et al., 2010). Yang et al. (2012) reported that sodium carbonate was a promising pretreatment for enhancing sugar yield of agricultural residues such as rice straw. During the process of green liquor or sodium carbonate pretreatment, all fermentable sugars are recovered in a single step of enzymatic hydrolysis, without production of fermentation inhibitors. Besides, the inorganic chemicals used are recovered and the dissolved organics including lignin are burned to produce energy, results a minimization of the operating costs.
As one of the world’s most widely grown crops, wheat straw is cultivated in over 115 nations under a wide range of environmental conditions. Over the past 100 years, the yields of wheat have been increased and annual global production of dry wheat in 2008 was estimated to be over 650 Tg (Farid et al., 2010). Wheat straw consists of two major parts, the leaf (leaf and sheath) and the stem. Cavity, parenchyma tissue, vascular bundles, sclerenchyma tissues and epidermis are presented from inner to outer of the stem. The chemical components are significantly different between the outer side and inner side of the
4
wheat straw stem, the inner side of the stem is rich in cellulose, and the outer side has more lignin. The contents of cellulose for both sides of the leaf sheath are almost the same (Yu et al., 2007). Besides, the dense layers of the cell wall give additional mechanical strength and higher tensile strength crystalline to the stem (Harper and Lynch, 1981). Since the characteristics of straw materials, such as more parenchyma cells, low lignin and high ash content, are rather different from those of wood, a mild pretreatment could be considered for an acceptable sugar yield in the following enzymatic hydrolysis process.
Based on the conception of GL process, sodium carbonate (SC) is an alternate pretreatment chemical for enhancing sugar yield of straw materials. The chemical can be recovered using a recovery boiler and its use does not require the supplement of sodium sulfate thereby simplifying the overall operation. On the other hand, problems of spent liquor evaporation and combustion in straw soda pulping mills can be effectively avoided since most silica retains in the pretreated solids. In this paper, the effects of sodium carbonate pretreatment at different alkali charge and temperature on the chemical compositions of wheat straw stem and leaf, as well as the sugar yield of enzymatic hydrolysis were comparatively investigated. As there is a large amount of leaf and sheath in the waste residues from straw handling yard, the results will demonstrate the possibility of using these residues as a free raw material for the production of bioethanol.
2. Materials and methods
2.1. Materials
5
Wheat straw (Triticum aestivum L.) was obtained from Jiangsu, China. The air dried straw was separated by hand into the stem and leaf (sheath included) carefully and cut to 3–5 cm in length. The samples were collected in sealed plastic bags and stored in a refrigerator at 4 °C. The main component of raw material was analyzed before pretreatment. Three enzymes, cellulase from Trichoderma reesei (NS-50013, 52.3 mg protein/mL, 84 FPU/mL), β-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, USA). All the chemicals were analytical grade and purchased from Nanjing Chemical Reagent Co., Ltd of China and used as received without further purification.
2.2. Sodium carbonate pretreatment
A rotary lab-scale cooking system with an electrically heated oil bath was used for the pretreatment. Ten 1 L-stainless steel bomb reactors with screw cap were contained in the cooking system. The pretreatment was carried out with a batch capacity of 80 g oven dry (od) wheat straw per bomb. The straw samples were directly subjected to pretreatment using sodium carbonate with different total titratable alkali (TTA) charge (6%, 8% and 10%, as Na2O). The ratio of liquor to straw was 6:1 (mL/g). The Na2CO3 concentration of the pretreatment liquors were 17.1, 22.8 and 28.5 g/L, and the corresponding pH-values were 11.19, 11.21 and 11.23, respectively. The materials were first impregnated with the pretreatment liquor at 80 °C for 30 min. Then the temperature was raised with the rate of
6
1.5 °C/min to the target temperature (120 °C, 130 °C and 140 °C). The pretreatment was immediately terminated while the designed temperature was reached. At the end of the pretreatment, the solids were collected and washed with hot water to remove residual chemicals and dissolved straw compounds. Pretreated solid yield, defined as the od weight of solid fraction divided by the od weight of original starting material, 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 KRK refiner (Φ 300 mm, 3000 rpm) was used for the defiberization of the pretreated wheat straw samples to produce pulp as substrates. Enzymatic hydrolysis of the substrates was carried out at substrate consistency of 5% (w/w) in sodium acetate buffer (pH 4.8) at 50°C using a shaking incubator at 180 r/min. An enzyme cocktail mixed by NS50013, NS-50010 and NS-50014 was used for enzymatic hydrolysis. The dosage of βglucosidase and xylanase supplementation constituted 30% of the volume of cellulase added (Lee et al., 2010). Enzyme cocktail loading was 10 and 20 FPU/g-cellulose based on cellulase activity. Tetracycline was charged at 40 μg/mL buffer as an antibiotic to inhibit microbial growth during the enzymatic hydrolysis. Enzymatic hydrolysis residue and hydrolysate was separated by centrifugation. Hydrolysates were sampled for monomeric sugar (glucose, xylose and arabinose) analysis.
2.4. Analytical methods
7
Cellulase activity in terms of “filter paper unit” (FPU) of cellulase 50013 was determined by the filter paper method using Whatman No. 1 filter paper as a standard substrate (Ghose, 1987). The enzyme protein content was determined using the Bradford method (Smith et al., 1985). Manufacturer specified activity of Novozymes 50014 and 50010 was directly used to calculate loading. The enzymatic hydrolysate was diluted 10 times and monomeric sugars in the hydrolysate were determined using a high performance liquid chromatography (Agilent 1200 Series, Santa Clara, CA) with refractive index detector (RID). A Bio Rad Aminex HPX-87H 20n exclusion column and a Cation-H refill cartridges (Bio-Rad Laboratories, Hercules, CA) were used as analytical column and guard column. The column temperature was 55 °C. A 5 m mol/L H2SO4 solution prepared with degassed super-purified deionized water was used as eluent at a flow rate of 0.6 mL/min. Aliquots (10 μL) were injected after passing through a 0.22 μm nylon syringe filter. 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 glucose, xylose and arabinose contents were corrected to anhydro units, i.e. glucan, xylan and arabinan for the calculation of sugar yield in enzymatic hydrolysate. Sugar yield in enzymatic hydrolysate was reported in weight percent of original glucose, xylose and total sugar in the original staring materials.
The content of benzene-ethanol extractives was determined according to Tappi Standard (T 204 cm-97). Lignin and carbohydrate contents of the raw and pretreated materials were analyzed using the NREL protocol (Sluiter et al., 2008). The Klason lignin content was
8
taken as the ash free residue after acid hydrolysis. The ash content was determined by combusting the samples in a furnace at 575 °C. The hydrolysate from this determination was retained for analysis of sugars and acid-soluble lignin (AS lignin). Sugars were determined by HPLC 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. To meet the requirement of column pH, 40 μL 50% (w/w) NaOH solution was injected per mL of hydrolysate sample prior to sugar analysis. Acid-soluble lignin was measured by absorbance at 205 nm in a 765 UV-VIS spectrometer. Each data point was the average of duplicate experiments.
3. Results and discussion
3.1. Chemical composition of wheat straw
The composition of wheat straw, wheat straw stem and wheat straw leaf (leaf and sheath included) used in this study is given in Table 1.
The composition of wheat straw, like other lignocellulosic biomass, is a complex mixture of cellulose, hemicellulose and lignin as three major components, and a small amount of extractives and ash. The benzene-ethanol extractives and ash in leaf were 6.5 and 11.3%, respectively, and those in stem were 4.6 and 7.6%, respectively. The higher content of extractives and ash in leaf is due to the higher content of epidermal cell, which is mainly composed by suberized cells and silica cells. Glucan and xylan are the two main
9
polysaccharides in wheat straw. The xylan content of stem was almost as the same as that of leaf, however, the glucan in stem was obviously higher than that in leaf. It means that compared with leaf, wheat straw stem contains more cellulose. Arabinan in stem was lower than that in leaf. The result indicates that, for wheat straw, the xylan structure of stem is different from that of leaf in some degree. The total lignin of stem was 23.0%, which was 4.9% higher than that of leaf (18.1%). Compared with stem, leaf had a higher content of Klason lignin and a lower content of acid-soluble lignin. The ratio of Klason lignin and acid-soluble lignin in stem was 9.2:1, while it was 5.7:1 in leaf. The sum of polysaccharides and lignin in stem was 86.0%, which was 10.1% higher than that in leaf.
3.2. Effect of sodium carbonate pretreatment on chemical composition of wheat straw
In this work, wheat straw, stem and leaf were directly subjected to pretreatment using sodium carbonate charge varied from 6% to 10% at 120 °C, 130 °C and 140 °C on the basis of bone dry materials. The effect of pretreatment temperature and TTA charge on the SC pretreated solid yield is shown in Fig. 1.
The pretreated solid yield of the different part of wheat straw under described conditions was approximately in the range of 60–90%. Random alkaline hydrolysis and secondary peeling reactions (Sjöström, 1993) did not occur in the sodium carbonate pretreatment as a result of the much lower pH-value of pretreatment liquor in comparison with that of the conventional kraft and soda cooking liquor.
10
The pretreated solid yield decreased with the increment of sodium carbonate charge and pretreatment temperature. As the alkalinity of sodium carbonate is weak, the increasing of pretreatment TTA charge from 6% to 10% at 120 °C only resulted in a small decrease of pretreated solid yield (ranged from 85.8% to 81.7%). However, when the pretreatment temperature was raised from 120 °C to 140 °C at 6% TTA charge, there was an obvious decrease of pretreated solid yield (ranged from 85.8% to 72.6%). It reveals that compared to the pretreatment temperature, the effect of TTA charge is not significant on the solid yields of wheat straw.
The SC pretreated solid yield of leaf was much lower than that of stem at the same temperature and TTA charge. It is mainly caused by the higher contents of benzene-ethanol extractives and ash in leaf in comparison with those in stem, as the benzene-ethanol extractives and the inorganic substances were easily dissolved at the pretreatment conditions. The yield of leaf pretreated with 10% TTA charge at 140 °C was 62.9%, which was the lowest among all the samples in this work. Harsh conditions during pretreatment resulted in a partial cellulose and hemicellulose degradation that could affect the following enzymatic hydrolysis (Lynd, 1996).
The effect of sodium carbonate pretreatment on the delignification of wheat straw was quantified by the reduction of lignin content in pretreated solids as a function of pretreatment temperature and TTA charge. As shown in Fig. 2, a good linear relationship can be observed for the solids pretreated by 6% to 10% TTA charge at 120 °C to 140 °C. At 140 °C, the lignin removal ranged from 35.1% at 6% TTA to a maximum of 44.7% at
11
10% TTA of the SC pretreated wheat straw, similar reductions in solid yields were attained irrespective of the leaf and stem. There was no obvious difference of the delignification selectivity among the wheat straw, stem and leaf samples pretreated with different TTA charge at 120 °C and 130 °C. The delignification selectivity of SC pretreated leaf became worse with the increase of pretreatment temperature. The pretreated solid yield dropped significantly at a same lignin removal level when the pretreatment temperature reached to 140 °C. Compared to the SC pretreated leaf in the same condition, the stem had a better delignification selectivity. For example, the pretreated solid yield of stem was 72.0% with a lignin removal of 39.1% when pretreated by 8% TTA at 140 °C; However, that of leaf was only 67.5% with a similar lignin content of 37.8% when pretreated by 6% TTA at 140 °C. It means that since straw lignin is easy to be removed even at a relatively low temperature, High pretreatment temperature does not help to improve delignification, but lowers the pretreated solid yield.
The percentage of each chemical component through SC pretreatment processes (TTA charge 6–10%, pretreatment temperature 120–140 °C) based on the original weight of different materials is presented in Fig. 3. It reveals that the losses of polysaccharides and lignin increase with the increment of TTA charge and pretreatment temperature. After the wheat straw was pretreated by 10% TTA charge at 140 °C, the degradation degree of glucan was approximately 10%, while xylan was over 28%. As briefly discussed previously, cellulose is more stable than hemicellulose under the SC pretreatment condition. Hemicellulose is relatively labile and dissolved in SC pretreatment at 140 °C.
12
Alkaline pretreatment partially solubilized the hemicellulose fraction and gave birth to a material enriched in cellulose (Silverstein et al., 2007; Varga et al., 2003).
Lignin is easily to be degraded under alkaline conditions. For example, the lignin removal of the SC pretreated wheat straw was 23.2% at 120 °C with 6% TTA charge, while the degree of polysaccharides degradation was only 1.2%. Meanwhile, the degrees of lignin and polysaccharides degradation at 140 °C with 10% TTA were 44.7 and 17.6%, respectively. It indicates that the degradation of lignin is higher than that of cellulose and hemicellulose during SC pretreatment, especially under a mild pretreatment condition. However, pretreatment under severe condition such as high temperature and high chemical charge resulted in an increasing degradation of polysaccharides. A high retention of sugar with a certain delignification is the principle point to an effective pretreatment for bioethanol production (Nigam, 2001; Tabka et al., 2006). The ash content of SC pretreated wheat straw was 4.7% at 120 °C with 6% TTA charge, which was only 1.3% higher than that at 140 °C with 10% TTA charge.
The comparison of main chemical components between SC pretreated leaf and stem under different pretreatment conditions are shown in Figs. 3(b) and 3(c). The degradation of carbohydrate was little for both stem and leaf when the samples were pretreated at 120 °C, and it became severe when the pretreatment temperature increased to 140 °C. More ash was retained in pretreated leaf solid in comparison with stem due to the high ash content in the original material. Lignin in leaf is easy to be removed even under a mild pretreatment condition. When the TTA charge ranges from 6% to 10% and the temperature ranged from
13
120 °C to 140 °C, the highest lignin removal of the SC pretreated leaf and stem were 58.1% and 44.7%, respectively. As lignin acts as a physical barrier to protect cellulose against microbial or chemical degradation (Mooney et al., 1998; Yu et al., 2011; Ding et al., 2012), the higher lignin content of the SC pretreated stem sample may influence the efficiency of the following enzymatic hydrolysis.
3.3. Effect of sodium carbonate pretreatment on the enzymatic hydrolysis of wheat straw
The SC pretreated solids were refined to produce pulp as substrate for enzymatic hydrolysis. The pulps were hydrolyzed by the enzyme mixture of cellulase, xylanase and β-glucosidase to evaluate the improvement of sugar yield. The xylanase used in the experiment was a little excessive. Supplementing the enzyme mixture with additional xylanase activity will improve saccharification of mildly treated samples containing higher amounts of xylan (Bura et al., 2009). The effects of sodium carbonate pretreatment temperature and TTA charge on sugar yield in enzymatic hydrolysate of SC pretreated wheat straw, stem and leaf are given in Tables 2 to 4.
The enzyme loadings of 10 FPU/g-cellulose and 20 FPU/g-cellulose were selected to evaluate the effect of sodium carbonate pretreatment on enzymatic hydrolysis. Total sugar in the tables is the sum of glucan, xylan and arabinan. The arabinan content is not listed since it is much lower than glucan and xylan. It is evident that the effect of enzyme dosage was more pronounced at 20 FPU/g-cellulose than at 10 FPU/g-cellulose. An obvious
14
increment of sugar yield could be observed for all SC pretreated samples when the enzyme loading was raised from 10 FPU/g-cellulose to 20 FPU/g-cellulose.
Table 2 shows that the total sugar yield of SC pretreated wheat straw pulp at 120 °C with 6% TTA charge was 57.6%, under an enzyme loading of 20 FPU/g-cellulose. The total sugar yield could be improved to 61.7% by increasing the TTA charge from 6% to 8%. There was no much difference between 8% and 10% TTA charge on the total sugar yield. Same situation was observed for the pulps pretreated at 130 °C and 140 °C. So it is clear that 8% TTA charge was a critical dosage for the pretreatment of wheat straw. The total sugar yield from wheat straw by SC pretreatment (8% TTA, 130 °C) and enzymatic hydrolysis (50 °C, pH 4.8, 48 h) using 20 FPU/g-cellulose was 64.8% (Table 2). It was the highest one among all pretreated wheat straw samples.
However, continuously increasing TTA charge from 6% to 10% of SC pretreated stem further improved the bioconversion of cellulose and hemicellulose to glucose and xylose (Table 3). In the condition of pretreatment temperature 130 °C and TTA charge 10%, the total sugar yield of the SC pretreated stem was 58.7%, which was the highest among all the pretreated stem samples. The glucan and xylan yield in hydrolysate were 56.2% and 62.1%, respectively in this condition. It could be calculated that about 39.1% of the cellulose and 23.4% of the hemicellulose were retained in the residue after enzymatic hydrolysis. The further pretreatment development is recommended to focus on the increase of cellulose accessibility, such as improving the TTA charge of SC pretreatment at 130 °C on wheat straw stem and the higher total sugar yield could be expected.
15
In contrast to the enzymatic hydrolysis efficiency of the stem, SC pretreatment on the leaf resulted in a larger increment in the total sugar yield of enzymatic hydrolysis (Tables 3 and 4). For example, at 130 °C with 10% TTA charge, the total sugar yield in hydrolysate of the SC pretreated leaf pulp was 73.1%, which was 14.4% higher than that of the pretreated stem pulp. The lignin removal of the SC pretreated leaf and stem under this condition were 50.0% and 31.5%, respectively. Lignin removal has been reported to improve cellulose accessibility effectively by creating pores and breaking the lignin-carbohydrate complex (Yang and Wyman, 2004). An increase in the surface area of the cellulose accessible to the cellulase would have a considerable effect on hydrolysis, as the enzyme requires contact with the substrate for hydrolysis to occur (Chandra et al., 2007). The enzymatic hydrolysis of leaf pulp pretreated with sodium carbonate (120 °C, 8% TTA charge) released 74.6% of total sugar, which was the highest among all the samples (Table 4). This research elucidates that straw leaf subjected by SC pretreatment can be easily converted to monomeric sugars during the enzymatic saccharification. The significant difference between the total sugar yield of leaf and stem indicates that the enzymatic hydrolysis efficiency of the wheat straw leaf is much better than that of stem.
The alkalinity of sodium carbonate is not strong enough to serve as an ideal pretreatment for wheat straw. In this work, the highest total sugar yields of SC pretreated wheat straw were 62.1%, 67.9% and 64.8%, respectively. It is lower than the sugar yield of wheat straw pretreated by sodium hydroxide or lime (Saha and Cotta, 2007; McIntosh and Vancov, 2011). Pedersen et al. (2010) found that the solid fraction was more susceptible to
16
enzymatic attack when wheat straw was pretreated by sodium hydroxide at pH 13. After hydrolyzed by a standard blend of Celluclast 1.5L and Novozyme 188, the glucose, xylose and total sugar yield obtained from enzymatic hydrolysis of the solid and liquid fraction were 76%, 92% and 80.1%, respectively. However, the total sugar yield became to 55.7% if the dissolved cellulose and hemicellulose in liquid fraction was not recovered. The total sugar yield of 64.8% is also lower than SC pretreated rice straw. Yang et al. (2012) reported that the sugar yield from rice straw by SC pretreatment (TTA 8%, 140 °C) and enzymatic hydrolysis (50 °C, pH 4.8, 48 h) using a cocktail of the same three enzymes at a loading of 20 FPU/g-cellulose was 71.7%. The higher portion of leaf and sheath in rice straw might be the key reason. As discussed earlier, SC pretreated leaf and sheath of wheat straw exhibits better efficiency of enzymatic saccharification than stem.
Soda pulping is regarded as a proven process to produce pulp using straw fiber as raw materials. Leaf and sheath should be removed as much as possible in straw handling yard to improve the quality of straw pulp. The results of this work show that straw handling rejects in a soda pulp mill is of great potential to be used as a cheap feedstock for the production of bioethanol using sodium carbonate as a pretreatment. A co-location bioethanol line using straw handling waste as raw materials could be considered in an operating soda pulp mill, as the pretreatment chemical, sodium carbonate can be recycled in the proven alkali recovery system. It will effectively cut the operation cost in bioethanol production.
3.4. Effect of lignin removal on enzymatic hydrolysis
17
The removal of lignin by SC pretreatment in this study may result in enhanced enzymatic hydrolysis by reducing the cellulase-lignin binding and increasing the accessibility of cellulose to the enzyme. It is reported that removal of 20–65% of the lignin in a sample is sufficient to increase the accessibility of the cellulose to enzymes (Ko et al., 2009). Fig. 4 illustrates the effect of lignin removal on total sugar yield of enzymatic hydrolysis at enzyme loadings of 10 FPU/g-cellulose and 20 FPU/g-cellulose. The total enzymatic hydrolysis sugar yield was increased with the lignin removal when the lignin removal was less than 30%. For most SC pretreated samples, the total sugar yield reached the highest when the lignin removal ranged from 30% to 40%. When the lignin removal of SC pretreated solids was more than 40%, enzymatic hydrolysis nearly leveled off. This is mainly due to the cleavage of aryl ether linkage of lignin accelerated by alkali during SC process, more lignin is fragmented and dissolved, and more surface area of carbohydrates is exposed. However, further delignification along with the decrement of pretreated solid yield influences the overall sugar yield of enzymatic hydrolysis of SC pretreated samples.
When comparing the total sugar yield to the same lignin basis, the SC pretreated leaf was more effective in enzymatic hydrolysis than stem. As shown in Fig. 4, when the lignin removal of SC pretreated stem pulp was 26.4% (130 °C, 8% TTA charge), the total sugar yield was only 56.3% after hydrolyzed with 20 FPU/g-cellulose. However, the total sugar yield of SC pretreated leaf pulp reached to 69.5% at a similar lignin content of 27.1% (120 °C, 6% TTA charge). In addition, there was a sharp increase of the lignin removal accompany with the decrease of the total sugar yield when the temperature reached to 140 °C. The highest total sugar yield of SC pretreated leaf solids (8% Na2CO3, 120 °C) was
18
74.5%. It was about 16% higher than the highest one of SC pretreated stem pulp (10% Na2CO3, 130 °C).
4. Conclusions
The chemical composition of wheat straw leaf is rather different from that of stem. Most of the polysaccharides are kept in the pretreated solids, and lignin in leaf is easy to be removed during SC pretreatment. The epretreated wheat straw leaf exhibits higher enzymatic hydrolysis efficiency in comparison with stem. The highest yield of total sugar from wheat straw leaf by SC pretreatment (8% TTA, 120 °C) and enzymatic hydrolysis (20 FPU/g-cellulose) was 74.5%, which was 16% higher than that from stem. The result provides a practical pretreatment pathway to produce bioethanol using the waste from straw handling yard.
Acknowledgements
The authors are grateful for the financial support of the National Basic Research Program of China (973 Program, Grant No. 2010CB732205), the National Natural Science Foundation of China (Grant No. 31070512), and the Priority Academic Program Development of Jiangsu Higher Education Institutions.
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Figure captions
Fig. 1. Effects of TTA charge and temperature on SC pretreated solid yield. Samples obtained at different pretreatment temperature from wheat straw, stem and leaf are expressed as W-T, S-T and L-T, in which T is pretreatment temperature. Fig. 2. Lignin removal as a function of sodium carbonate pretreated solid yield. Samples obtained at different pretreatment temperature from wheat straw, stem and leaf are expressed as W-T, S-T and L-T, in which T is pretreatment temperature. The three data points on each line are corresponding to the different TTA charge of pretreatment: 6%, 8% and 10% (from left to right). Fig. 3. Retention of various components in sodium carbonate pretreated wheat straw (a), stem (b) and leaf (c). Fig. 4. Effect of lignin removal on total sugar yield of enzymatic hydrolysis at cellulase loading of 10 FPU/g-cellulose (a) and 20 FPU/g-cellulose (b). Samples obtained at different pretreatment temperature from wheat straw, stem and leaf are expressed as W-T, S-T and L-T, in which T is pretreatment temperature. The three data points on each line are corresponding to the different TTA charge of pretreatment: 6%, 8% and 10% (from left to right).
25
Fig. 1.
100
80
70
60
50 6 8 TTA (%) 10 L-140
L-130
L-120
W-140
W-130
Samples
26
W-120
S-140
S-130
S-120
Pretreated solid yield (%)
90
Fig. 2. 70 L-120 L-130 L-140
Lignin removal (%)
60 50
W-120 W-130 W-140
S-120 S-130 S-140
40 30 20 10 0 60
65
70
75
80
85
Pretreated solid yield (%)
27
90
95
Fig. 3. 100 90 80 Ccontent (%)
70 60 50 40 30 20 10 0 Raw
6%
8%
10%
120˚C
6%
8%
10%
6%
130˚C Wheat straw
8%
10%
140˚C
(a) 100 90 80 Content (%)
70 60 50 40 30 20 10 0 Raw
6%
8%
10%
6%
120˚C
8% 130˚C
Stem
(b)
28
10%
6%
8% 140˚C
10%
100 90 80
Content (%)
70 60 50 40 30 20 10 0 Raw
6%
8%
10%
6%
120˚C
Glucan AS lignin
8%
10%
130˚C Leaf Arabinan Extractive
Xylan Ash
(c)
29
6%
8%
10%
140˚C
Klason lignin
Fig. 4. 80
Total sugar yield (%)
70 60 50 40 L-120 W-120 S-120
30
L-130 W-130 S-130
L-140 W-140 S-140
20 0
20
40
60
80
Lignin removal in pretreatment (%)
(a) 80
Total sugar yield (%)
70 60 50 40 L-120 W-120 S-120
30
L-130 W-130 S-130
L-140 W-140 S-140
20 0
20
40
60
Lignin removal in pretreatment (%)
(b)
30
80
Table 1 The main chemical components of wheat straw samples (w/w, %). Standard deviations are given between brackets. Benzeneethanol Extractives Wheat straw 5.0 (0.4) Stem 4.6 (0.2) Leaf 6.5 (0.4) Samples
Lignin AcidKlason soluble 19.0 (0.3) 2.5 (0.0) 20.8 (0.1) 2.2 (0.0) 15.4 (0.0) 2.7 (0.0)
Sugar Total
Glucan
Ash Xylan
Arabinan Total
21.5 (0.3) 37.9 (0.5) 19.9 (0.2) 2.9 (0.2) 60.7 (0.5) 8.0 (0.6) 23.0 (0.1) 40.4 (0.1) 20.1 (0.2) 2.5 (0.3) 63.0 (0.6) 7.6 (0.2) 18.1 (0.0) 34.9 (0.2) 19.8 (0.2) 3.0 (0.2) 57.7 (0.1) 11.3 (0.7)
31
Table 2 Effects of pretreatment temperature and TTA charge on sugar yield in enzymatic hydrolysate of SC pretreated wheat straw. Standard deviations are given between brackets. Cellulase loading 10 FPU/g-cellulose Cellulase loading 20 FPU/g-cellulose
Temp. (°C)
TTA charge in pretreatment (%)
Glucan (%) Xylan (%) Total a (%) Glucan (%) Xylan (%) Total (%)
120
6
46.7 (0.1)
45.1 (0.3)
47.5 (0.1)
57.9 (0.3)
55.5 (1.6)
57.6 (0.6)
8
47.7 (0.1)
47.8 (3.2)
51.7 (2.1)
59.2 (0.2)
61.9 (0.1)
61.7 (0.7)
10
44.5 (0.8)
54.9 (1.0)
50.1 (0.6)
58.9 (0.2)
62.3 (0.5)
62.3 (0.3)
6
46.3 (1.7)
48.8 (1.9)
47.3 (1.7)
55.7 (2.9)
56.4 (1.6)
55.9 (2.3)
8
47.1 (0.2)
56.7 (0.7)
51.2 (0.9)
62.1 (0.7)
67.9 (2.0)
64.8 (0.1)
10
46.1 (1.4)
56.7 (2.1)
51.1 (1.5)
61.2 (0.3)
65.9 (0.7)
63.6 (0.1)
6
42.5 (0.6)
45.0 (2.0)
45.7 (1.3)
54.9 (1.1)
52.1 (2.3)
56.6 (1.3)
8
43.5 (0.6)
47.9 (0.5)
47.1 (0.9)
57.8 (1.6)
54.7 (1.9)
59.4 (1.9)
10
42.8 (0.1)
45.3 (1.0)
46.0 (0.2)
56.8 (1.1)
52.3 (0.4)
57.6 (0.8)
130
140
a
Total sugar is the sum of glucan, xylan and arabinan. Data of arabinan are not listed due to
the low content.
32
Table 3 Effects of pretreatment temperature and TTA charge on sugar yield in enzymatic hydrolysate of SC pretreated wheat straw stem. Standard deviations are given between brackets. Cellulase loading 10 FPU/g-cellulose Cellulase loading 20 FPU/g-cellulose
Temp. (°C)
TTA charge in pretreatment (%)
Glucan (%) Xylan (%) Total (%)
Glucan (%) Xylan (%) Total (%)
120
6
38.1 (1.6)
41.8 (1.2)
39.2 (0.6)
46.4 (0.5)
48.1 (0.4)
47.8 (0.8)
8
33.8 (1.1)
51.6 (1.4)
40.6 (0.1)
48.8 (0.5)
59.6 (1.2)
53.2 (0.8)
10
33.7 (0.6)
53.6 (1.2)
41.0 (0.1)
51.2 (0.0)
62.0 (0.0)
55.7 (0.0)
6
35.3 (0.5)
49.4 (0.4)
40.5 (0.5)
53.8 (1.1)
60.2 (0.0)
56.0 (0.6)
8
36.4 (0.3)
50.9 (0.1)
41.9 (0.2)
54.3 (0.7)
59.7 (0.1)
56.3 (0.5)
10
36.6 (0.6)
51.4 (1.8)
42.1 (0.9)
56.2 (1.0)
62.1 (2.1)
58.7 (1.0)
6
35.5 (0.5)
44.7 (0.8)
38.7 (0.1)
52.2 (0.6)
51.2 (0.1)
52.1 (0.4)
8
35.1 (0.1)
47.4 (0.3)
39.7 (0.0)
54.1 (0.7)
55.8 (1.1)
54.7 (0.7)
10
34.5 (0.6)
46.3 (1.3)
38.7 (0.1)
52.5 (0.9)
56.8 (0.2)
54.0 (0.2)
130
140
33
Table 4 Effects of pretreatment temperature and TTA charge on sugar yield in enzymatic hydrolysate of SC pretreated wheat straw leaf. Standard deviations are given between brackets. Cellulase loading 10 FPU/g-cellulose Cellulase loading 20 FPU/g-cellulose
Temp. (°C)
TTA charge in pretreatment (%)
Glucan (%) Xylan (%) Total (%)
Glucan (%) Xylan (%) Total (%)
120
6
65.7 (0.8)
51.8 (0.6)
60.4 (0.5)
75.7 (0.4)
60.0 (0.3)
69.6 (0.5)
8
64.6 (0.4)
59.5 (0.3)
62.8 (0.3)
78.7 (0.1)
67.9 (0.6)
74.6 (0.3)
10
63.3 (0.0)
56.8 (0.2)
61.0 (0.0)
79.5 (0.4)
68.2 (3.0)
74.4 (1.2)
6
65.4 (0.8)
53.3 (0.2)
60.5 (0.4)
79.0 (0.5)
63.1 (1.3)
72.3 (0.7)
8
65.3 (0.6)
57.1 (0.9)
61.7 (0.8)
79.9 (0.4)
65.0 (0.7)
73.8 (0.3)
10
64.6 (0.9)
55.1 (0.9)
60.1 (0.9)
79.3 (1.1)
64.0 (0.2)
73.1 (0.8)
6
61.9 (1.2)
46.3 (2.1)
55.3 (1.4)
70.5 (1.7)
52.0 (1.6)
65.0 (1.5)
8
61.0 (0.6)
47.9 (0.2)
58.7 (0.9)
70.7 (1.7)
53.3 (0.3)
67.9 (1.0)
10
59.8 (0.7)
46.8 (0.9)
56.8 (0.6)
70.8 (1.0)
54.7 (2.9)
67.6 (1.4)
130
140
34
The highlights of the paper are: • Sodium carbonate (SC) was used as a mild alkaline pretreatment on wheat straw; • Chemical composition of SC pretreated wheat straw stem and leaf was compared; • Lignin in leaf was easier to be removed than that in stem; • Enzymatic hydrolysis of SC pretreated wheat straw stem and leaf was compared; • Sugar yield of pretreated wheat straw leaf is much higher than that of stem.
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