Process intensification effect of ball milling on the hydrothermal pretreatment for corn straw enzymolysis

Energy Conversion and Management 101 (2015) 481–488

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Process intensification effect of ball milling on the hydrothermal pretreatment for corn straw enzymolysis Zhengqiu Yuan a,b, Jinxing Long a, Tiejun Wang a,⇑, Riyang Shu a,b, Qi Zhang a, Longlong Ma a a b

Key Laboratory of Renewable Energy, Guangzhou Institute of Energy Conversion, Chinese Academy of Science, Guangzhou 510640, PR China University of Chinese Academy of Science, Beijing 100049, PR China

a r t i c l e

i n f o

Article history: Received 20 March 2015 Accepted 26 May 2015 Available online 16 June 2015 Keywords: Ball milling Cellulose Delignification Glucose Cellulase Bioconversion

a b s t r a c t Enhancement of the cellulose accessibility is significant for biomass enzymatic hydrolysis. Here, we reported an efficient combined pretreatment for corn straw enzymolysis using ball milling and dilute acid hydrothermal method (a mixture solvent of H2O/ethanol/sulfuric acid/hydrogen peroxide liquid). The process intensification effect of ball milling on the pretreatment of the corn straw was studied through the comparative characterization of the physical–chemical properties of the raw and pretreated corn straw using FT-IR, BET, XRD, SEM, and HPLC analysis. The effect of the pretreatment temperature was also investigated. Furthermore, various pretreatment methods were compared as well. Moreover, the pretreatment performance was measured by enzymolysis. The results showed that ball milling had a significant process intensification effect on the corn straw enzymolysis. The glucose concentration was dramatically increased from 0.41 to 13.86 mg mL1 after the combined treatment of ball milling and hydrothermal. The efficient removal of lignin and hemicellulose and the enlargement of the surface area were considered to be responsible for this significant increase based on the intensive analysis on the main components and the physical–chemical properties of the raw and pretreated corn straw. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction The plants produce the most abundant and the cheapest cellulosic biomass resource on the earth through photosynthesis process. It is attracting more and more attention due to the increasingly prominent contradiction between the supplies and demands of fossil resource and the serious environmental issues [1–4]. Corn straw is a typical cellulosic biomass and is widely used as a promising raw material for bulks of liquid fuels and chemicals [5,6]. However, the cellulose of this material is generally existed as long, oriented microfibrils by the b-1,4-linked bonds in the cell wall, resulting in high hydrophobic and crystalline [7]. Furthermore, it is interconnected with the other two components (lignin and hemicellulose) by hydrogen bond and chemical bond that contributes to biomass recalcitrance [8]. Therefore, pretreatment is necessary to destroy the lignin seal and disrupt the crystal structure of cellulose for the increase accessibility of cellulase enzymes during bioconversion process [9,10]. Generally, the pretreatment methods for cellulosic biomass mainly contain physical, chemical and biological methods, which are devoted to increase the accessibility of enzyme catalyst, ⇑ Corresponding author. Tel.: +86 20 87057673; fax: +86 20 87057789. E-mail address: [email protected] (T. Wang). http://dx.doi.org/10.1016/j.enconman.2015.05.057 0196-8904/Ó 2015 Elsevier Ltd. All rights reserved.

enhancing directly the utilization of cellulosic biomass [11,12]. Dilute acid pretreatment method is an efficient chemical method for cellulosic biomass [13], but, its major disadvantage is the concomitants of sugar degradation products like hydroxymethyl furfural (HMF) and furfural (FF), which are toxic for enzymolysis process [14]. Hydrogen peroxide has been reported to be efficient reagent for the removing or degrading lignin and hemicellulose [15], because of its effect with the hydrogen bond and relatively stability in acidic medium [16]. It contributes to destroy the crystal structure of cellulose and disrupt the force among three components. Organosolv pretreatment method can efficiently extract lignin from biomass to reduce the reconstituted lignin recovered on the material surface, which hinders the hydrolysis and limits the recycling of cellulase enzymes [17]. In this way, ethanol can provide an outlet for this swamp since its excellent dissolving capacity for lignin. However, the efficiency of this pretreatment is still improvable because of the intensive inner structure and the complex component connects in the cellulosic biomass. According to the previous studies [18,19], ball milling can efficient reduce the particle size and loosen the inner structure of the biomass, which has a great potential to the promotion of the pretreatment efficiency for biomass enzymolysis. Based on the kind knowledge of abovementioned process, we proposed here an efficient pretreatment process for corn straw

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enzymolysis using the combined ball milling and chemical (ball milling before chemical pretreatment, BMC) method. In which, ball milling was used as physical process for the reduction of particle size and cellulose crystallinity, and increase of the surface area which enhanced enzyme loading. And then, a liquid mixture containing sulfuric acid, hydrogen peroxide, ethanol and water was used as chemical pretreatment to remove the lignin and hemicellulose, the major obstacles for cellulose enzymatic hydrolysis. The advantage of this combined pretreated method (BMC) was studied through the compared investigation of various pretreatment methods, such as single ball milling (BM), single chemical hydrothermal (chemical) and combined pretreatment of chemical hydrothermal before ball milling (CBM). We also examined the intensification mechanism of the ball milling on the corn straw pretreatment via compared investigation on the changes of the chemical composition, structure, and physical–chemical property using component analysis, fourier transform infrared spectra (FT-IR), Brunauer–Emmett–Teller surface area measurement (BET), X-ray diffraction (XRD), and scanning electron micrograph (SEM). Furthermore, we also investigated the enzymolysis performances of the pretreated corn straw from various conditions. The results demonstrated that ball milling had a significant promotion effect on the corn straw pretreatment. In which, 34 times of glucose yield could be obtained by enzymatic hydrolysis compared with that from raw corn straw.

times. Ball milling with chemical (BMC) and chemical with ball milling (CBM) pretreatments were the combination of different sequence with the two pretreatment methods mentioned above. 2.3. Raw and pretreated corn straw characterization The compositions of the raw and pretreated corn straw were obtained by NREL method. Summarily, 0.5 g corn straw sample was added to 200 mL ethanol for Soxhlet extraction in water-bath. Then the residue was air-dried and transferred into 25 mL centrifugal tube containing 3 mL 72% sulfuric acid in water-bath at 30 °C with stirring. The mixture after concentrated acidic hydrolysis was transferred to 150 mL triangle flask, and 84 mL distilled water was added to dilute the acidic concentration to 4%. 10 mL sugar recovery standard liquid was added to another 150 mL triangle flask to calculate the sugar recovery rate. All flasks were placed in the autoclave at 121 °C for 45 min. The final mixture was filtered with sand-core funnels, and the filtrate volume (L) was measured. 5 mL filtrate was neutralized by 8% sodium hydroxide solution and then diluted to 10 mL. The sugar concentration was determined by High Performance Liquid Chromatography (HPLC) to ensure the amount of cellulose and hemicellulose.

Monosaccharide concentration :

C x ¼ C HPLC;x 

Dilution ration Rav e;sugar

Average simple sugar recovery rate: 2. Material and methods Rav e;sugar ¼

2.1. Raw materials Raw corn straw was collected from Liaoning province, China. The stems were air-dried and size-reduced to 40–60 mesh, and washed with distilled water to clean the biomass surface prior to oven-dried at 80 °C until a constant weight. Corn straw powder composition (w/w) was determined by the National Renewable Energy Laboratory (NREL) analytical method [20]. The results showed that it was composed of cellulose as glucose of 44.50%, hemicellulose as xylose and arabinose of 19.69%, acid insoluble lignin of 25.35%, ash of 1.26%, moisture of 5.84% and others of 3.36%. Celluclast 1.5L (cellulase) and Novozym 188 (b-glucosidase) were purchased from Sigma–Aldrich. The enzyme activities were determined to be 80 FPU mL1 (expressed as micromoles of glucose produced per minute, with filter paper as a substrate) and 246.4 CBU mL1 (expressed as micromoles of glucose that is converted to glucose per minute, with cellobiose as a substrate) for Celluclast 1.5L and Novozyme 188, respectively [21]. Other reagents were analytical grade and supplied by Aladdin Co., Ltd. 2.2. Pretreatment processes for corn straw Physical pretreatment (ball milling) was performed on a QM-3SP04 planetary ball milling apparatus (Nanjing University Instrument Factory, Jiangsu China), which was equipped with four 100 mL ZrO2 jars (ZrO2 spheres diameter u = 5, 7, 10 mm). After evenly filled by 40 g oven-dried corn straw (40–60 mesh), the ZrO2 jars were rotated at 500 rpm for 20 h. Chemical pretreatment was defined as liquid-phase pretreatment. 3.0 g dried corn straw was added into a 100 mL Teflon lined autoclave with 60 g liquid mixture containing 75% ethanol, 0.9% peroxide, 1.96% sulfuric acid and 22.14% water (w/w). The mixture was treated at designated temperatures of 100–150 °C for 120 min. After the reaction was finished, the reactor was cooled down to room temperature using flowing water. And then, the mixture was centrifuged at 6000 rpm for 10 min, and the precipitate was oven-dried at 80 °C after washing with distilled water for three

monosaccharide concentration before dilute acidic hydrolysis monosaccharide concentration after dilute acidic hydrolysis

Cellulose amount :

/C ¼ C glu  87  103 L  0:9=m0

Hemicellulose amount :

/H

¼ ðC xyl þ C ara Þ  87  103 L  0:88=m0 The residue from the filtered process was washed with hot distilled water until it was neutral, then oven-dried at 105 °C (weighing m1), and transferred to the crucible which was placed in a muffle furnace at 550 °C for 6 h (weighing m2).

Lignin amount :

/L ¼ ðm1  m2 Þ=m0

where m0 was the primary weight of material; L was the final volume of the hydrolyzate; 0.9 and 0.88 are hydro correction for glucose and xylose, respectively; hemicellulose amount was based on the sum of xylose and arabinose because xylose and arabinose were the main composition in hemicellulose. The metal elements in the ash were determined by inductively coupled plasma-atomic emission spectrometry (ICP-AES, OPTMIMA 8000). FT-IR spectra were obtained on a Nicolet iS50 FT-IR spectrophotometer (Thermo Scientific, USA). The instrument was worked with a mercury cadmium telluride (MCT) detector and the spectrum was recorded in the range of 4000–400 cm1. BET was used to determine the surface and total volume of samples by N2 adsorption isotherm at 77 K using a surface analyzer. Scanning Electronic Microscopy (SEM, HITACHI S-4800 instrument) was involved to observe the characteristic changes of treated samples. X-ray diffractometer (PANalyticalX’pert Pro MPD) was used to measure the samples’ crystallinity with Cu Ka radiation at 40 kV and 300 mA. The samples were scanned in the 2h range of 5–80° at a rate of 2° per minute. The crystallinity index (CrI) was calculated by the following method [22].

CrI ¼ ðI002  Iam Þ  100=I002 where I002 was the intensity of the 002 peak at 2h = 22.5° and Iam was the intensity of 2h = 18.7°.

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2.4. Enzymatic hydrolysis of the pretreated corn straw In a 150 mL triangular flask, 1.0 g pretreated sample was suspended in 30 mL citric acid-sodium citrate buffer (50 mmol L1, pH = 4.8). After that, Celluclast 1.5L (cellulase, 50 FPU g1 substrate) and Novozyme 188 (b-glucosidase, 30 CBU g1 substrate) were added to investigate enzymatic hydrolysis of pretreated corn straw. The mixture was then incubated at 55 °C and 130 rpm for 48 h in a water-bath shaker. 2.5. Product analysis

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was introduced (Fig. 1b). Moreover, Fig. 1 showed that all of the three monosaccharides in liquid-phase were significantly increased during the BMC process (Fig. 1b) than the single thermal–chemical process (Fig. 1a). Physical ball milling provided the extrusion stress for destroying the structure of corn straw, resulting in less particle size and looser structure. Therefore, the removal of the hemicellulose was easier. 3.2. Delignification of corn straw

The concentration of glucose, xylose and cellobiose after hydrolysis were determined by HPLC analysis. Some details were as following. The hydrolysis solution was first centrifuged at 6000 rpm for 10 min, and the upper-layer supernatant was then neutralized and injected into an 87H column (6.5  300 mm) operating on Waters 2695S Controller machine with a Waters e2695 RID detector. The working condition of HPLC analysis as follow: volume temperature 45 °C, detector temperature 50 °C, and the mobile phase was 0.005 mol L1 sulfuric acid solution with the flow rate of 0.55 mL min1.

Lignin is another barrier in cellulosic biomass enzymatic hydrolysis [24]. The removal degree of this aromatic polymer from corn straw with or without ball milling was shown in Fig. 2. During the single chemical pretreatment process (without ball milling), 46.42% lignin could be extracted from corn straw at 100 °C, and then the lignin extraction efficiency increased gradually at higher pretreatment temperatures with the highest value of 65.86% at 150 °C. However, with the combined pretreatment of BMC process, the delignification was significantly enhanced. For instance, 56.21% of lignin was extracted from corn straw at 100 °C and the highest value of 70.65% at 150 °C, which was higher than many reported biomass pretreatment processes [25,26].

3. Results and discussion

3.3. Characterization of the pretreated corn straw

3.1. Hemicellulose removal

3.3.1. Cellulose content The component of the original and pretreated corn straws were determined by NREL analytical method [20], and the results were listed in Table 1. Cellulose, hemicellulose and lignin contents in raw corn straw were 44.50%, 19.69% and 25.35%, respectively. However, when it was treated by the dilute acid with or without ball milling, the components of the corn straw were significantly changed. The cellulose content was obviously increased with the significant decrease of lignin and hemicellulose (Table 1). For example, 75.97% and 80.50% of the pretreated corn straw were composed of cellulose after single chemical and combined pretreated process respectively. It should be noted that, compared with the single chemical process, the combined process (BMC method) was more efficient. Higher cellulose content and the degree of lignin removal (80.99% for combined and 75.42% for single chemical process) were shown when a ball milling process was presented, which accords well with the above results as shown in Fig. 2. The facts that the intensive hydrogen bond and the connection between lignin and the carbohydrate in corn straw was weakened [27], and the structure of the corn straw was loosened, were considered to be ascribed to the more flexible for delignification. This conclusion can be further confirmed by the contents change

Hemicellulose is a main barrier for cellulose enzymolysis [23]. Therefore, the removal of the hemicellulose was first investigated in the chemical and BMC process. The results shown in Fig. 1 demonstrated that the xylose began to release at the temperature of 100 °C, which illustrated that xylan was firstly depolymerized. With the increase of the reaction temperature, the hemicellulose hydrolysis was enhanced. For example, xylose concentration was 0.35 mg mL1 at 100 °C, then 1.38 mg mL1 at 120 °C, and finally 2.70 mg mL1 at 150 °C without ball milling (Fig. 1a). Fig. 1a also showed that the amorphous cellulose from the periphery of the cellulose elementary fibril was also hydrolyzed when the temperature was more than 120 °C. And almost 0.08 mg mL1 of the glucose was identified at 120 °C. However, when the raw corn straw was ball milled before the chemical treatment (BMC method), the onset hydrolysis temperature of cellulose was significantly decreased (from 120 to 110 °C, Fig. 1b). Furthermore, the removal of the hemicellulose was also significantly promoted. For instance, xylose yield was 0.87 mg mL1 at 110 °C in chemical pretreatment process (Fig. 1a), however, it dramatically increased to 1.16 mg mL1 at the same temperature when the ball milling

Fig. 1. Removal of the hemicellulose of corn straw. (a) Without ball milling and (b) with ball milling. Conditions: corn straw 3.0 g; mixture liquid 60 g; 120 min.

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be extracted by ethanol and H2O with the acid catalyst [30,31]. Therefore, the lignin content in the pretreated corn straw was decreased with the increase of pretreatment temperature from 100 °C to 120 °C (Table 2 and Table S2). However, lignin is aromatic polymer and can repolymerize at the temperature from 120 °C to 140 °C [32], so lignin content was slight increased at this temperature range. However, the depolymerization of this aromatic polymer was enhanced at elevated temperature of 150 °C, resulting in the declination of lignin content. It should be noted that the lignin content of the pretreated corn straw by combined method was far lower than it from the single chemical method (24.58% for single chemical method and 19.01% for BMC method) at the same temperature. Therefore, above mentioned results (Tables 1 and 2 and Table S2) demonstrated clearly that BMC pretreatment has an obvious advantage for both the hemicellulose and lignin removal.

Fig. 2. Delignification of corn straw with various methods. Conditions: corn straw 3.0 g; mixture liquid 60 g; 120 min.

Table 1 Chemical composition of raw and pretreated corn straw.a

Cellulose (%) Hemicellulose (%) Lignin (%) Ash (%)b Others (%)c Hemicellulose removal (%) Lignin removal (%) Ash removal (%) Others removal (%)

Raw

Chemical

BMC

44.50 19.69 25.53 1.43 8.85 – – – –

75.97 11.18 6.23 1.89 4.43 43.22 75.42 65.29 73.02

80.50 12.59 4.82 2.14 0.95 36.06 80.99 84.61 94.5

a The contents of cellulose, hemicellulose and lignin were determined by NREL analytical method. Samples of residue obtained from selected temperature at 120 °C. b Measured by the combustion TG from 50 to 900 °C with 10 °C min1 under oxygen. c Evaluated by the conservation of mass.

of ash and others (which was generally composed of protein and lipid [28]). As shown in Table 1, more than 94.5% of protein and lipid were removed under the combined pretreatment of BMC with the 84.61% weight loss of ash. Furthermore, ICP-AES analysis result (Table S1) showed that the appropriate contents of iron and magnesium ions can improve the cellulase activity [29]. Temperature also had a substantial effect on the hemicellulose removal and delignification (Table 2 and Table S2). With the increase of the reaction temperature, the cellulose content of the pretreated corn straw was significantly increased with both the single chemical and combined methods. The highest cellulose content of 97.73% and 95.18% could be obtained when the pretreatment temperature increased to 150 °C. Simultaneously, the hemicellulose content gradually decreased at the elevating temperature, and it was completely removed at 150 °C. Generally, lignin could

3.3.2. Surface morphology change of the original and pretreated corn straw Efficient enzymatic hydrolysis depends on the accessibility of cellulose system. The removal of lignin and hemicellulose can enhance the accessibility of cellulose system, but cellulase activities are always carried out on the surface of the solid cellulose [33]. Therefore, SEM was used to determine the characteristic changes in morphology during various pretreatments. Fig. 3a showed the SEM image of an untreated vascular bundle with a diameter of around 150 lm, which had a tight and smooth surface. The cells were linked together. Ball milling completely destroyed the skeleton of cells to small particle, so available surface area was created (Fig. 3b). The corn straw by chemical pretreatment became loose and depicted partial separation of fiber with the obvious collapsing of the main cell framework, which can be interpreted by the fact that hemicellulose and lignin were removed by the liquid-phase extraction. However, the main skeleton of the corn straw could be seen clearly (Fig. 3c). The SEM images of the corn straw after combined pretreatment of ball milling and chemical methods (CBM and BMC methods) showed a significant difference (Fig. 3d and e). Generally, in a chemical treatment process, the lignin–carbohydrate complex (the link between lignin and carbohydrate) was firstly broken [7]. Therefore, the hemicellulose and lignin were partially removed leaving a loose and lamellar structure. This loose corn straw was more flexible for particle size reduction under ball milling. However, the small particles were easy to gather together during the ball milling process as shown in Fig. 3d. Combination process of BMC pretreatment avoided this disadvantage. Small size particles formed by ball milling dispersed in the liquid-phase to remove the lignin and hemicellulose so that cellulose fiber can be fully exposed. Accordingly, the reduction of particle size and removal of lignin and hemicellulose can increase accessibility of cellulose system, and enhance the contact between cellulases and cellulose fiber, which was favorable for enzymatic hydrolysis. 3.3.3. FT-IR spectra FT-IR spectroscopy analysis was used to determine the constituent and chemical structure changes of corn straw by different

Table 2 Chemical composition of BMC pretreated corn straw at different temperature.a

a

Pretreated temp. (°C)

Cellulose (%)

Hemicellulose (%)

Lignin (%)

Hemicellulose removal (%)

Lignin removal (%)

100 110 120 130 140 150

65.11 75.62 80.5 85.16 84.33 97.73

17.73 13.35 12.59 5.47 4.39 0

11.71 5.86 4.82 5.56 9.8 2.16

9.95 32.20 36.06 72.22 77.70 100.00

53.81 76.88 80.99 78.07 61.34 91.84

The contents of cellulose, hemicellulose and lignin were determined by NREL method.

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Fig. 3. SEM morphologies of raw and pretreated corn straw. (a) Corn straw, (b) ball milled corn straw, (c) chemical pretreated corn straw, (d) CBM pretreated corn straw and (e) BMC pretreated corn straw. Samples of residue obtained from selected temperature at 120 °C.

pretreatment methods. The bands listed in Table S3 were assigned according to the reported literatures [34,35]. The infrared bands at 3390 cm1 was caused by AOH stretching vibrations. Absorbance at 1255 and 1731 cm1 were assigned to the characteristic absorbance of xylan in hemicellulose. Absorption peaks at about 3390, 2900, 1375, and 1165 cm1 were attributed to cellulose. Lignin showed the characteristic absorption bands at 2900, 1600–1500, 1423, 1314 and 830–750 cm1 [36,37]. FT-IR spectra of raw and pretreated corn straw by different pretreatments at 120 °C were shown in Fig. 4. The spectrum of the raw material contained the characteristic absorption peaks of cellulose, hemicellulose and lignin (Fig. 4a). The infrared absorption at 1110 cm1 was weakened when the corn straw was treated by single ball milling (Fig. 4b). It indicated that the OAH association between cellulose and hemicellulose was destroyed by ball milling pretreatment [27]. For the sample with chemical pretreatment (Fig. 4c), the disappeared absorbencies at 1731, 1633, 1602, 1512, 1462, 1255, 834 cm1 implied that almost all of hemicellulose was removed and partial loss of aromatic ring in lignin. FT-IR spectrum of the combined pretreated corn straw

Fig. 4. The dynamics changes of FT-IR spectra of various pretreated corn straw at 120 °C. (a) Raw, (b) ball milling, (c) chemical, (d) CBM and (e) BMC.

(Fig. 4d and e) was different with them of all the above samples As shown in Fig. 4e, the characteristic infrared absorption strength of the hemicellulose, which generally showed absorption at 1255 and 1731 cm1, was remarkably decreased. It suggested the efficient removal of the hemicellulose, which accords well with the results as shown in Tables 1 and 2. As shown in Fig. 4e, the characteristic absorption of the benzene ring structure was significantly weakened after the combined treatment, which clearly demonstrated the efficient delignification under the cooperative effect of ball milling and chemical treatment. In contrast, the enhanced absorption at 3390, 2900, 1642, 1060 cm1, which was generally considered to be the characteristic infrared absorption of the cellulose [35], clearly demonstrated that the remained corn straw was mainly composed of cellulose. Therefore, the FT-IR spectra of corn straw with various pretreatments further confirmed that ball milling had a significant enhancement effect for the removal of hemicellulose and lignin when it was combined with chemical pretreatment. Fig. S4 showed that the FT-IR spectra of pretreated corn straw by BMC pretreatment at various temperatures ranged from 100 to 150 °C. The absorbencies at 1255 and 1731 cm1, which represent the CAO and C@O stretching vibrations of the acetyl ester unit of hemicellulose, was reduced gradually with the increase of temperature and completed disappeared at 150 °C, implying the efficient hemicellulose degradation. This conclusion accords well with the above composition results (Table 1), where the hemicellulose began to be degraded at 100 °C and completely removed at 150 °C. The bands of 1614, 1507 and 1462 cm1, which represented the C@C stretching from the aromatic ring, was observed and increased with the elevated temperature when it was lower than 140 °C, and then they slight decreased at higher temperature. It indicated the efficient delignification as shown in Fig. 2. 3.3.4. X-ray diffraction and surface area analysis Crystallinity is an essential factor for enzymatic hydrolysis. Different pretreatments can change the hydrogen bond in cellulose fiber [38]. As shown in Table 3, the original corn straw had a crystallinity of 55.4%. However, when it was treated by dilute acid hydrothermal process, the crystallinity of the residual solid sharply increased to 71.1%. It was considered that the removal of lignin and hemicellulose (Figs. 1 and 2, Table 1) which are amorphous, is

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Table 3 Crystallinity and surface area of corn straw from various pretreatments.a

a

Material

Crystallinity

Surface area (m2 g1)

Raw Chemical Ball milling CBM BMC

55.4 71.1 Amorphous Amorphous 44.05

0.944 3.142 2.631 1.894 2.947

Samples of residue obtained from selected temperature at 120 °C.

responsible for this crystalline degree increase. Ball milling is an efficient pretreatment method for cellulose enzymolysis, because both the hydrogen bond and the crystallinity of the cellulose can be broken. Therefore, the pretreated corn straw from the single ball milling and CBM process were amorphous (Table 3). However, it should be noted that, the crystallinity of the pretreated corn straw was 44.05% when the raw material was suffered a BMC process (Table 3). In this process, the corn straw was ball milled to be amorphous at first, and then the removal of hemicellulose and lignin by the chemical pretreatment lead to the crystallinity increase. This conclusion can also be confirmed by the crystallinity of the pretreated corn straw under various temperatures (Table 4). As discussed above, the delignification and the removal of hemicellulose were more efficient at elevated temperature (Figs. 1 and 2, and Table 1), leaving higher content of the crystal cellulose. Therefore, the crystallinity increased proportionally with the increasing temperature. The increase of CrI implied that the amorphous portion in corn straw such as hemicellulose and lignin were indeed removed, which increased the accessibility of the crystalline cellulose for cellulase. In addition, nitrogen isotherms were adopted to compare surface area change of the corn straw by different pretreatment methods. As expected, specific surface area was significantly increased with the pretreatment. For example, the surface of the raw material was sharply increased from 0.944 to 3.142 m2 g1 when it was treated by the chemical method. This significant increase could be attributed to the fact that the efficient removal of the lignin resulting in small pore in the cellulose frame [37] as exhibited by SEM images (Fig. 3c). After ball milling, the cellulose frame was collapsed with the disappearance of the small pore, and the small particles were easy to gather together during the ball milling process (Fig. 3d). Therefore, the surface area of the corn straw was decreased to 1.894 m2 g1 (Table 3). Extrusion stress of ball milling created tensile physical forces to break corn straw into the fragmented tissues, and shear stress during the chemical pretreatment created tensile chemical forces that destroyed the structure of the corn straw by the extraction of lignin and hemicellulose. Therefore, the sample after ball milling prior to hydrothermal effect had a specific area of 2.947 m2 g1, which was slight lower than it from the single chemical treatment but was higher than other (Table 3). However, it should be noted that the tight cellulose frame of the corn straw which was far more recalcitrant for conversion [39,40], was disappeared (Fig. 3e) with the intensification effect of ball milling on the chemical pretreatment. Furthermore,

Table 4 Crystallinity and surface area of corn straw from BMC pretreatment at different temperatures. 2

Temperature (°C)

Crystallinity

Surface area (m g

100 110 120 130 140 150

34.8 40.45 44.05 53.41 57.52 62.1

1.863 2.384 2.947 2.287 0.771 0.372

1

)

the crystallization of cellulose was sharply decreased as verified by X-ray diffraction (XRD) analysis results (Table 3). Therefore, the corn straw after combined pretreatment of BMC was more flexible for the enzyme accessibility of cellulose. Table 4 showed the effect of the pretreatment temperature on the crystallinity and the surface area of the BMC corn straw samples. Just as the discussion shown above, the crystallinity increased gradually with the elevated temperature. It is attributed to the efficient hemicellulose removal and delignification (Figs. 1 and 2, Tables 1 and 2) and the hydrolysis of the amorphous cellulose fiber. It can also be seen that the surface area of the pretreated corn straw sample was increased when the reaction temperature was less than 120 °C, and then, it was declined with the increasing elevation of the reaction temperature (Table 4). As shown in Fig. 1, the cellulose of BMC corn straw sample was degraded at 120 °C, so the surface area increase at low temperature could be attributed to the increasingly loose cellulose structure. At high temperature, the amorphous cellulose was hydrolyzed and the cellulose frame caused by the partially removal of hemicellulose and lignin was destroyed (Fig. 3e). Therefore, the surface of the BMC corn straw was decreased when the temperature was more than 120 °C. 3.4. Enzymatic saccharification Table 5 summarized the enzymatic hydrolysis results of raw and pretreated corn straw from different pretreatment methods pretreated at 120 °C. Glucose and xylose yield was determined by HPLC chromatogram. As shown in Table 5, the yield of monosaccharide was significantly affected by the pretreatment method. After 48 h of enzymatic hydrolysis, the glucose yield of raw corn straw was 0.41 mg mL1, and only 2.63% glucan in cellulose was converted to glucose. Ball milling pretreated corn straw achieved the glucose yield of 4.04 mg mL1 by enzymatic hydrolysis, and the glucan conversion increased to 25.87%. It was considered that ball milling can only increase the surface area (Table 3), but could not destroy the linkage between cellulose and hemicellulose and lignin (Fig. 4), which negatively affected the enzymatic hydrolysis by cross-linking with cellulose. Single chemical pretreatment was inefficient, where only 1.73 mg mL1 of glucose yield could be obtained under the optimized condition (Table 5 and Fig. 5). It was considered that the high crystallinity of the pretreated corn straw (Table 3) and tight cellulose frame (Fig. 3c) were the main obstacles for the efficient cellulose enzymolysis. After ball milling, the cellulose frame was collapsed, therefore, the yield of glucose was sharply increased to 6.74 mg mL1. The intensification effect of the ball milling was more obvious when the raw material was hydrothermal treatment after ball milling. At the same enzymolysis condition, the highest glucose yield of 13.86 mg mL1 could be achieved, which was 34 times of it without pretreatment and 8 times of the glucose with single chemical pretreatment (Table 5). The efficient removal of lignin and hemicellulose (80.99% of the lignin and 36.06% of hemicellulose, Figs. 1, 2 and 4), decrease of the

Table 5 Sugar yields of enzymatic hydrolysis of the raw and pretreated corn straw.a Sample Raw Ball milling Chemical CBM BMC

Glucose (mg mL1)

Xylose (mg mL1)

Glucan conversion (%)

0.41 4.04

0.00 2.31

2.63 25.87

1.73 6.74 13.86

1.03 2.31 1.95

6.49 25.28 49.07

a Samples of residue obtained from selected temperature at 120 °C. Conditions: 1 g substrate in 30 mL buffer; 50 FPU of Celluclast 1.5L and 30 CBU of Novozyme 188 at pH 4.8, 55 °C, and 130 rpm for 48 h.

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Fig. 5. Sugar yields of enzymatic hydrolysis of the different pretreated corn straw. (a) Chemical, (b) CBM and (c) BMC. Conditions: 1 g substrate in 30 mL buffer; 50 FPU of Celluclast 1.5L and 30 CBU of Novozyme 188 at pH 4.8, 55 °C, and 130 rpm for 48 h.

crystallinity, and the increase of the specific surface area (Table 3) were ascribed to the significant enzymolysis enhancement. This intensification effect is also more obvious than the reported corn straw combined pretreatment using ozonolysis and ball milling (the sugar yield was about 9 times of that without pretreatment) [41] and our previous process of ultrafine grinding [42]. The saccharification of the pretreated corn straw samples (chemical, CBM and BMC) with various temperatures was also investigated. Fig. 5 demonstrated that the pretreated temperature has a substantial effect on the enzymolysis of the corn straw. Both the glucan conversion and the glucose yield suffered a first increase and then decrease with the peak value at 120 °C (the optimized pretreatment temperature). Fig. 5 also demonstrated that the sugar yield from the enzymatic hydrolysis of the BMC corn straw (Fig. 5c) was far higher than that from single chemical (Fig. 5a) and CBM sample (Fig. 5b) at each pretreatment temperature. For instance, more than 11.5 mg mL1 glucose yield could be obtained for the BMC sample when the pretreatment temperature was more than 110 °C, whereas the glucose yield was no more than 2 mg mL1 (Fig. 5a) and 7 mg mL1 (Fig. 5b) for single chemical and CBM corn straws respectively even under the optimized pretreatment temperature. It further confirmed the abovementioned conclusion that ball milling has a substantial intensification effect for the corn straw pretreatment when it was conducted before a chemical process.

milling followed by chemical pretreatment (H2O/ethane/sulfuric acid/hydrogen peroxide liquid mixture), the raw material was more flexible for enzymolysis. In this process, ball milling destroyed the physical structure of corn straw, then the downstream chemical pretreatment removed lignin and hemicellulose to enhance the accessibility of cellulose. Under the optimized conditions, more than 13.86 mg mL1 glucose concentration could be achieved. This glucose yield was 34 times of it without pretreatment and 8 times of it with single chemical pretreatment. Detail analysis demonstrated that the efficient delignification and hemicellulose removal, decrease of the crystallinity degree, and increase of the surface area were responsible for this corn straw enzymolysis enhancement. And thus, the presented method for corn straw pretreatment would have a great potential on the biomass utilization. Acknowledgments The authors gratefully acknowledge the financial support of the National High Technology Research and Development Program of China (‘‘863’’ Program: 2012AA101806), the National Natural Science Foundation of China (No. 51306191), and the National Key Technology R&D Program (No. 2014BAD02B01).

Appendix A. Supplementary material 4. Conclusion Ball milling showed a significant intensification effect on the corn straw enzymolysis. With the combined treatment of ball

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.enconman.2015. 05.057.

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