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Bioresource Technology 99 (2008) 54745479

Organosolv pretreatment by crude glycerol from oleochemicals industry for enzymatic hydrolysis of wheat straw
Fubao Sun, Hongzhang Chen
*
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, P.O. Box 353, Beijing 100080, China Received 27 September 2007; received in revised form 1 November 2007; accepted 1 November 2007 Available online 20 February 2008

Abstract In order to defray the cost of biodiesel production, the ensuing work was to further investigate utilization of the crude glycerol (CG) from oleochemicals industry in the atmospheric autocatalytic organosolv pretreatment (AAOP) to enhance enzymatic hydrolysis. The AAOPCG enabled wheat straw to achieve with reasonable enzymatic hydrolysis yields, reaching P75% for the wet substrate and 663% for the dried. Lipophilic compounds from the CG formed pitch deposition on the ber, which was responsible for low delignication (630%) and also troublesome in practical operation. Pitch deposits itself had no signicant role on enzymatic hydrolysis. A striking nding of the lignin recondensation and/or lignincarbohydrate complex helped explain why dried pretreated wheat straw had a low enzymatic hydrolysis yield. The CG was suitable for the AAOP to enhance enzymatic hydrolysis of lignocellulosic biomass. But it was advisable to remove lipophilic compounds from crude glycerol before utilization. 2007 Elsevier Ltd. All rights reserved.
Keywords: Crude glycerol; Atmospheric autocatalytic organosolv pretreatment; Enzymatic hydrolysis; Lignocellulosic biomass; Oleochemical industry

1. Introduction Renewable lignocellulosic biomass, as a promising alternative for the limited crude oil, can be utilized to produce biofuels and biochemicals. In order to make these biobased products more cost-competitive with fossil-derived conventional commodities, pretreatment and enzymatic hydrolysis of lignocellulosic biomass has become two key processes involved in the bioconversion of carbohydrates to inexpensive reducing sugars (Sun and Cheng, 2002). And the recovery of cellulose and its susceptibility to enzymatic attack are generally taken into the technologyeconomic consideration of the pretreatment process (Nguyen and Saddler, 1991; Mosier et al., 2005). Considering that hemicelluloses and lignin are the main limiting factors of enzymatic hydrolysis, many research eorts in the pretreat*

Corresponding author. Tel.: +86 10 82627067; fax: +86 10 82627071. E-mail address: hzchen@home.ipe.ac.cn (H. Chen).

ment have been made to remove them as much as possible and simultaneously keep enough cellulose intact, such as dilute acid (Sun and Cheng, 2005; Cara et al., 2007), alkaline solution (Kim and Holtzapple, 2005; Zhao et al., 2007), steam explosion (Chen et al., 2002; Chen and Liu, 2007; Ohgren et al., 2007), and liquid hot water (Laser et al., 2002; Liu and Wyman, 2005). However, these present pretreatment techniques are still encountered with some eciency-high, cost-eective and environment-friendly problem. Organosolv pulping, as a promising pretreatment strategy, has attracted much attention and has demonstrated the potential utilization in lignocellulosic biomass (Jimenez et al., 2002; Pan and Sano, 2005; Pan et al., 2005). But the low-boiling-point organosolv pulping was restricted to the laboratory or pilot scale for several reasons, including the risk from high pressure operation and the highly volatile and ammable solvent. Meanwhile, mainly due to the high commercial price of solvent, so far limited research

0960-8524/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.11.001

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interests have been focused on the high-boiling-point organosolv pulping process (Rezayati-Charani et al., 2006; Rodriguez et al., 2007; Jimenez et al., 2007). But glycerol, as a high-boiling-point organic solvent, is presently the main byproduct of oleochemicals industry, as high as 10% of the total biodiesel output. Since the soaring petroleum price has made the oleochemicals industry, especially biodiesel production, more ourishing, glycerol products are driven rapidly to oversupply and have created a drastic glut on the market (de Guzman, 2005a). And the cheap glycerol, whose price even declined to about 10 cents per pound, has slaughtered on the whole EU and North America market (de Guzman, 2005b). Although there is extensive utilization of high purity glycerol (>99.0%) in food, cosmetic and pharmaceutical industry, it is technically dicult and costly to obtain the pure glycerol from biodiesel production (Pachauri and He, 2006). It is, therefore, desirable to explore novel, economic utilization of crude glycerol in order to escape the collapse of glycerol price and further defray the cost of biodiesel production (ODriscoll, 2007). Little research has been done on alternative utilizations of glycerol as a cooking solvent, treating the lignocellulosic biomass. The only sample was that of Kucuk and Demir bas (1993) and Demirbas (1998), who took the lead in conducting on the delignication of wood by aqueous glycerol. Research results indicated that aqueous glycerol (72% consistency) organosolv pulping could give a high lignin remove from wood biomass. Our earlier work of atmospheric autocatalytic organosolv pretreatment (AAOP) has not only conrmed this, but also found that the pretreatment can greatly enhance enzymatic hydrolysis of lignocellulosic biomass. But the cooking solvent used was industrial glycerol (Sun and Chen, 2007) or its 70% aqueous glycerol liquor which would be reported elsewhere. Since crude glycerol (CG) from oleochemicals industry has a consistency of 7080%, it is more interesting economically if the crude glycerol can be used directly in organosolv pretreatment of lignocellulosic biomass to improve enzymatic hydrolysis. If possible, it would avoid further processing of the crude glycerol from oleochemicals industry, such as desalination, distillation and discolouration. Based on this, our present work was proposed to investigate the utilization of CG byproducts from oleochemicals industry in treating wheat straw to enhance enzymatic hydrolysis. And some explanations were given, based on the SEM, to the striking phenomena occurring in pretreatment and enzymatic hydrolysis. 2. Experimental 2.1. Materials Air-dried wheat straw was collected in Henan Province, China. It was manually cut into pieces for an approximately 20 mm in length, dried to constant weight at

60 C and then stored in polyethylene plastic containers. The original wheat straw and insoluble solid ber fractions were dried at 60 C to constant weight for dried weights determination. Their main components were analyzed by a modied sequential gravimetric method (Datta, 1981; Robertson and van Soest, 1981). The average main components (%, w/w) of starting materials, as determined by three replicate analyses, were as follows: cellulose, 43.2%; hemicelluloses, 31.7% and lignin, 17.3%. All experiments were performed in duplicate under the same condition and average values were reported. The standard deviations were less than 6%. Industrial glycerol (IG) of Commercial grade (95% purity) was purchased as the byproduct of sebacic acid from Hengshui Jinghua Chemical Plant, Hebei Province, China. It was diluted to concentration of 70% (w/w) in the experiment; Crude glycerol from sebacic acid production (CGSAP) was purchased as the above. Its main composition was as follows (% wt): 4050% glycerol, 78% salt, 2530% water content, 24% non-glycerol organic substance and 12% oil. It was condensed to 70% for utilization; Crude glycerol from biodiesel production (CGBP) was kindly presented by Applied Chemistry Institute of Tsinghua University. The main composition was as follows (% wt): 70% glycerol, 58% salt, 1520% water content, 46% non-glycerol organic substance and 0.30.5% oil. 2.2. The AAOPCG process Our previous work of the AAOP had found the controlled experimental conditions, at 220 C for 3 h with the liquidsolid ratio of 200/10, which would be reported elsewhere. Based on it, 10 g dry wheat straw in a typical run was suspended in 200 g, 70% dierent-type aqueous glycerol liquor. After a heat to 220 C, the temperature was kept for 3 h and then began to descend. The slurry was cooled to 120130 C, added slowly with 200 mL recycling industrial glycerol liquor (app. 40% consistency) and stirred vigorously. The experiment was performed under reux. Heating was by an electric heater and temperature was indicated by a thermometer (Demirbas, 1998). After thorough disintegration, the insoluble solid ber fraction was separated by ltration through G3 glass lter and washed twice with 400 mL recycling industrial glycerol liquor. After a wash for three times with 600 mL tap water, the pretreated ber was then divided into two parts. One part was dried to determine the yield of pretreatment and chemical components as well as the brous structure, and the other was conserved in a sealed bag at 5 C, the ber fraction to be used for further enzymatic hydrolysis. In order to get rid of the lipophilic compounds from dierent-type glycerol, deposited on the ber, some dry solid ber (app. 1.0 g) was taken with 250 mL petroleum ether in a Soxhlet apparatus to extract for 6 h. Each sample was performed in duplicate. The standard deviation was less than 4%.

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Yields of pretreatment and components (% the original)

2.3. Enzymatic hydrolysis of the resulting ber fractions Just like our previous reports (Jin and Chen, 2006; Sun and Chen, 2007), a mixture of Penicillium decumbens JUA10 cellulase and b-glucosidase, supplied by Ningxia Cellulase Preparation Plant, China, was used to detect enzymatic convertibility of the ber fraction to reducing sugar. Filter paper activity (FPA) of Cellulase and b-glucosidase activity were determined as 110 FPU mL1 and 42 IU mL1, according to the methods of Ghose (1987) and Kubicek (1982), respectively. Each individual sample (approximately 0.5 g dry weight) of wet or dried solid ber fraction from the pretreatment process was suspended quickly in some (0.2 mol L1, pH 4.8) acetate buer solution in a 100 mL ask and diluted to 25.5 g, which achieved with 2% solids (w/w), to reduce end-product inhibition. The slurry was then supplemented with cellulase enzymes of 44 FPU g1 dry matter and bglucosidase of 16.8 IU g1 dry matter. Enzymatic hydrolysis was performed at 180 rpm in a rotary shaker at 50 C for 48 h (Sun and Chen, 2007). The slurry was sampled periodically to determine reducing sugar for according to Millers method (1959). Conversion eciency of the original wheat straw, based on reducing sugar produced by pretreatment and enzymatic hydrolysis, was designated as follows: Reducing sugar yield (%) = (g in reducing sugar) (100 g in original wheat straw)1. Each sample was performed in duplicate, with the average value reported. The standard deviation was less than 3.2%. 2.4. Structural observation on ber surface by SEM The microscope pictures were taken by a eld emission scanning electron microscope (SEM, JSM-6700F, JEOL, Japan) after the samples were sputtered with a thick layer of gold spread uniformly from all sides under two dierent angles. 3. Result and discussion 3.1. Chemical composition of wheat straw pretreated by dierent glycerol Fig. 1 illustrates the yield of pretreatment and main components from wheat straw pretreated by dierent types of glycerol liquor. The glycerol type included IG, CGSAP and CGBP. As shown in Fig. 1, compared with 64% of the IG, the pulp yields led by the crude glycerol, CGSAP and CGBP, increased 12% and 5%, respectively. But their cellulose began a slight degradation, reaching 96% and 94% of the original, correspondingly. The data indicated that crude glycerol seemed to have a poor ability of hemicelluloses and lignin removal. On the other hand, the CGSAP only removed 45% of the original hemicelluloses and 27% lignin, far lower than 68% and 64% of that by IG, respectively. Although the CGBP gave 75% hemicelluloses removal, the delignication by it was extremely low and

110 100 90 80 70 60 50 40 30 20 10 0 IG CGSAP CGBP

Glycerol type Pulp Cellulose Hemicellulsoe Lignin

Fig. 1. Chemical composition of wheat straw pretreated in 70% dierenttype glycerol liquor at 220 C for 3 h. The dierent-type glycerol included industrial glycerol (IG), crude glycerol from sebacic acid production (CGSAP) and crude glycerol from biodiesel production (CGBP).

only 18.5%. This showed that the crude glycerol pretreatment could not eectively remove the lignin from lignocellulosic biomass, though the rened glycerol presented the superior performance in delignication (Demirbas, 1998; Sun and Chen, 2007). The direct utilization of crude glycerol was unaccepted for lignin removal. What caused this possibly resulted from the impurities in crude glycerol, including salt, oil and non-glycerol organic substance, for industrial glycerol was processed from crude glycerol in oleochemicals industry. In order to explore the impurities from crude glycerol, the dried insoluble solid ber from dierent-type glycerol pretreatment was extracted by petroleum ether for 6 h. The result was found that the ber obtained from IG pretreatment had nearly no petroleum ether extracts. But the lipophilic extracts reached 10% (dry wt) of the insoluble solid ber from CGSAP pretreatment and 28% from CGBP, respectively. This indicated that some lipophilic compounds from crude glycerol survived the cooking and washes process, and nally deposited on the resulting ber. In order to further conrm the pitch deposition formed by lipophilic compounds from crude glycerol, SEM images were produced on the crude glycerol pretreated ber before petroleum ether extraction (images not shown). Compared with IG pretreated wheat straw, there appeared some large numbers of nubbly deposits on the CGSAP and CGBP pretreated ber. The observation indicated that nearly no pitch deposition occurred on the IG pretreated ber. But there were many lipophilic deposits on the CGSAP and CGBP pretreated ber. These lipophilic compounds deposited alone or with other components such as inorganic salts present in the pretreatment process (Gutierrez and del Ro,

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2005). The observations were consistent with the above petroleum ether extraction analysis. They strongly supported that lipophilic compounds survived the cooking and wash process and nally formed the so-called pitch deposition on the ber. Similar information on pitch deposition has been reported extensively in (non-) wood paper pulping (del Ro et al., 1999; Gutierrez et al., 2001; Gutierrez and del Ro, 2005). These literatures have reported that during conventional pulping many lipophilic compounds in parenchyma cells and softwood resin canals can survive the pulping process and form colloidal pitch deposition. The pitch deposits are often dicult to remove in the washing stage and exist in a way of sticky deposits on the ber pulp and process equipments, so they often result in blockages and even a shutdown of operations. Moreover, the pitch deposition tends to produce phenomena of resin obstruction and lignin passivation, by decreasing the chemical transfer rate and the permeability of ber in the aqueous phase, which are extraordinarily adverse to pulp bleaching. Consequently, the pitch deposition has long become a seri ous trouble in the pulp and paper industry (del Ro et al., 1999). Therefore, the poor lignin removal led by crude glycerol originated from the lipophilic compounds contained it, which hindered the lignin dissociation in cooking process and/or its washing-o in wash process. 3.2. Enzymatic hydrolysis of the crude glycerol pretreated wheat straw The wet insoluble solid ber fraction, after the above three-type glycerol pretreatment, was directly used as a substrate for enzymatic hydrolysis. The prole of enzymatic hydrolysis of glycerol pretreated wheat straw is shown in Fig. 2. After 48 h, the enzymatic hydrolysis yield
95 90 85

of CGSAP and CGBP pretreated wheat straws reached 75% and 77%, respectively, while that was $90% for the IG pretreated ber. From the point of feedstock-to-sugar production, the conversion eciency of wheat straw, after IG, CGSAP and CGBP pretreatment, reached 47%, 44% and 37%, respectively. The data indicated that the enzymatic hydrolysis yield and conversion eciency of wheat straw were improved by CGSAP and CGBP pretreatment. But they were signicantly lower than those by IG pretreatment, which resulted partly from the residual lignin content. Besides, the conversion eciency of wheat straw by CGBP pretreatment was signicantly lower than that by CGSAP, while enzymatic hydrolysis yield of the former was higher. This was because CGBP pretreated ber had a slight cellulose degradation and serious hemicelluloses removal, which resulted in the low reducing sugar yield even if a high enzymatic hydrolysis yield. 3.3. Eect of pitch deposits on enzymatic hydrolysis of CG pretreated ber Since such high lipophilic compounds survived the whole pretreatment process and deposited on the pretreated ber, it was worthwhile to explore the eect of pitch deposits on enzymatic hydrolysis of CG pretreated ber. In order to avoid the extra inuence of dierent moisture in wet ber before and after petroleum ether extraction, the dried ber was taken as a substrate in the experiment. The samples (0.5 g) of dried crude glycerol pretreated wheat straw before and after petroleum ether extraction (PEE) were taken, respectively, as the substrates and suspended in the acetate buer (25 mL) for enzymatic hydrolysis of 48 h. The prole of their enzymatic hydrolysis is described in Fig. 3. As shown in Fig. 3, enzymatic hydrolysis yields of CGSAP and CGBP pretreated wheat straws before PEE reached $63% and $49%, respectively, whereas $65% and $51% after PEE, correspondingly. It
75 70

Enzymatic hydrolysis yield (%)

Enzymatic hydrolysis yield (%)

80 75 70 65 60 55 50 45 40 35 30 0 8 16 24 32 40 By IG By CGSAP By CGBP 48

65 60 55 50 45 40 35 30 25 0 8 16 24 32 CGSAP CGSAP-PEE CGBP CGBP-PEE 40 48

Time (h)
Fig. 2. Enzymatic hydrolysis of the above pretreated wheat straw ber. Enzymatic hydrolysis was carried out at 50 C, 180 rpm in pH 4.8, 0.2 mol L1 acetate buer with enzyme additions of 44 FPU g1 and 16.8 IU g1 dry matter.

Time (h)
Fig. 3. Enzymatic hydrolysis of the dried CGSAP and CGBP pretreated wheat straws before and after petroleum ether extraction (PEE). Enzymatic hydrolysis was carried out as the above.

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was suggested that the pitch deposits had no signicant eect on enzymatic hydrolysis of crude glycerol pretreated lignocellulosic biomass. By comparison with enzymatic hydrolysis of the wet and dried CG pretreated wheat straws (Figs. 2 and 3), it was found that enzymatic hydrolysis yield of the dried pretreated ber decreased drastically. Moreover, enzymatic hydrolysis yield of the dried CGSAP pretreated ber was signicantly higher than that from CGBP pretreatment, which was not uniform to the enzymatic hydrolysis yield displayed in Fig. 2. The nding meant that the drying process of wet pretreated ber probably directed the structural alteration of ber towards aggravating inhibition on enzymatic hydrolysis. And the resistance, arising from structural alteration, strengthened with an increase of residual lignin content. 3.4. SEM observation on the outer structure of pretreated ber In order to verify the above deduction, structural observation was tried to examine the status of dried pretreated ber. SEM images were made on CGBPPEE pretreated ber surface (images not shown). With a preliminary structural observation, some speckles were found on the surface of CG pretreated ber. By the further ultra-structural observation, the small speckle was made commonly of some granules (app. 0.10.2 lm in diameter) and/or an agglomeration (app. 0.20.4 lm in length). Zhang (2003) in her work of aqueous ethanol autocatalytic organosolv pulping also noticed some spherical granules on the ber surface. She argued that the spherical sediment on the Chinese-alpine-rush ber surface was a lignin scrap, easy to form a granule, which had been removed by cooking and left behind at wash. Yang et al. (2005) have, further, demonstrated that the make-up in the corncob was a xylanlignin complex by ultra-structural observation (magnication 8000). Moreover, images supplied on the micro-morphology of xylanlignin complex were highly similar to ours. Therefore, it was inferred that the impurity on the CG pretreated ber surface was a lignin recondensation and/or lignincarbohydrate complex (LCC) with the super-molecular structure. And the components, the granule and agglomeration, were a lignin scrap fraction and a polysaccharide fraction, respectively. Many factors, such as the lignocellulosic variety and processing method, resulted in heterogeneity of the LCC structure, which manifested the dierent characteristics (Lawoko, 2005). Although the lignin was a key limiting factor of enzymatic hydrolysis (Sun and Cheng, 2002), there was no clear correlation between the lignin content and enzymatic hydrolysis yield of lignocellulosic biomass (Palonen, 2004). Palonen (2004) has also concluded that the location and chemical/physical structure of lignin aect the enzymatic hydrolysis more than the absolute amount of lignin. It was, therefore, postulated that the lignin recondensation and/or LCC, formed on dried pretreated ber,

intensied the inhibitive role of residual lignin on enzymatic hydrolysis. Judging from the lignin recondensation and/or LCC deposited on the CG pretreated ber surface, some hemicelluloses and lignin were dissociated with cellulose in cooking process. Owing to the pitch deposition, they left behind and precipitated on the ber at wash stage. Meanwhile, the residual lignin probably did not present a serious inhibitory role on enzymatic hydrolysis because of the sole precipitation on the ber. But the drying process led to the structural alteration of lignin and carbohydrates, forming a lignin recondensation and/or LCC, which aggravated the inhibitory role of the residual lignin. Furthermore, the inhibitive role increased with high lignin content. This helped make clear that the oven-dried CG pretreated wheat straw achieved with a much lower enzymatic hydrolysis yield than the wet one. It also explained why the dried CGSAP pretreated ber had a higher enzymatic hydrolysis yield than the CGBP pretreated. 4. Conclusions The AAOP, using the crude glycerol from oleochemicals industry, was suitable for enhancing enzymatic hydrolysis. But lipophilic compounds from crude glycerol formed the pitch deposition in the pretreatment process. It was advisable to get rid of them from crude glycerol before utilization. A striking nding was that some residual lignin and carbohydrates formed the lignin recondensation and/or LCC during the drying process of CG pretreated ber. The drying directed structural modication of residual lignin and carbohydrates towards aggravating the inhibition on enzymatic hydrolysis. Such the inhibitive role was probably responsible for low enzymatic hydrolysis yield of dried ber. Acknowledgements This work was nancially supported by National Basic Research Program of China (973 Project, No. 2004CB719700) and National Key Project of Scientic and Technical Supporting Programs Funded by Ministry of Science & Technology of China during the 11th Fiveyear Plan Period (No. 2006BAD07A07). References
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