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Feb 8, 2008 - BACKGROUND: The oversupply of cheap glycerol by the oleochemicals industry together with problems occurring in low-boiling-point ...
Journal of Chemical Technology and Biotechnology

J Chem Technol Biotechnol 83:707–714 (2008)

Comparison of atmospheric aqueous glycerol and steam explosion pretreatments of wheat straw for enhanced enzymatic hydrolysis Fubao Sun1,2 and Hongzhang Chen1,2∗ 1 National

Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100080, China 2 Graduate School of the Chinese Academy of Sciences, Beijing 100080, China

Abstract BACKGROUND: The oversupply of cheap glycerol by the oleochemicals industry together with problems occurring in low-boiling-point organosolv pretreatments, has generated an interest in the use of glycerol in the organosolv pretreatment of lignocellulosic biomass. Atmospheric aqueous glycerol autocatalytic organosolv pretreatment (AAGAOP) is a promising strategy that can effectively enhance enzymatic hydrolysis of lignocellulosic biomass. As a cost-effective technique, steam explosion pretreatment (SEP) is being adopted in industrial applications. Accordingly, work has been carried out to investigate how AAGAOP enhanced enzymatic hydrolysis of lignocellulosic biomass compares with the SEP method. RESULTS: Under controlled laboratory conditions, based on ≥90% cellulose recovery, AAGAOP removed ≥60% hemicellulose and ≥60% lignin from wheat straw while SEP led to ∼80% hemicellulose and 10% lignin removal. Enzymatic hydrolysis yields of AAGAOP and SEP reached ∼90% and ∼70%, respectively. Physical-chemical structural characterization by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FT-IR), helped explain the above results. The two methods gave priority to dissociating the guaiacyl lignin and had a relatively small effect on syringyl units. However, AAGAOP exhibited a superior performance. CONCLUSION: The two methods enhanced the enzymatic hydrolysis of lignocellulosic biomass by removing and/or altering physical-chemical structural impediments. The AAGAOP technique, with some special advantages, was more effective than SEP in enhancing the recovery and enzymatic digestibility of cellulose.  2008 Society of Chemical Industry

Keywords: atmospheric aqueous glycerol autocatalytic organosolv pretreatment; steam explosion; enzymatic hydrolysis; lignocellulosic biomass; wheat straw; biodiesel

INTRODUCTION Renewable lignocellulosic biomass, as a promising alternative to limited crude oil, can be utilized to produce biofuels and biochemicals. To make these bio-based products more cost-competitive with fossilderived conventional commodities, pretreatment and enzymatic hydrolysis of lignocellulosic biomass have become two key processes in the production of inexpensive reducing sugars.1 The recovery of cellulose, and its susceptibility to enzymatic attack are generally taken into account when evaluating the technology/economy of pretreatment processes.2 Because hemicellulose and lignin are key limiting factors, much research interest has been focused on removing as much of them as possible in pretreatments such as

the acid/alkaline method,3,4 wet oxidation5,6 and liquid hot water.7 However, these present pretreatment techniques have some intractable problems concerning high efficiency, cost-effectiveness and environmentfriendly aspects.8 Organosolv pulping is a promising pretreatment strategy that has attracted much attention and demonstrated potential utilization with lignocellulosic biomass.9 – 11 However, low-boiling-point organic solvents such as methanol, ethanol and acetone, are restricted to laboratory- or pilot-scale for several reasons, including the risk from the high pressure operation and the highly volatile and flammable solvent. Meanwhile, mainly due to the high cost of solvent, little research has focused on high-boiling-point organosolv pretreatment processes.12 – 14

∗ Correspondence to: Hongzhang Chen, National Key Laboratory of Biochemical Engineering, Chinese Academy of Sciences, PO Box 353, Beijing 100080, China E-mail: [email protected] (Received 22 October 2007; revised version received 14 November 2007; accepted 15 November 2007) Published online 8 February 2008; DOI: 10.1002/jctb.1860

 2008 Society of Chemical Industry. J Chem Technol Biotechnol 0268–2575/2008/$30.00

F Sun, HZ Chen

Glycerol, a high-boiling-point organic solvent, is presently the main byproduct of the oleochemicals industry, and can be as high as 10% of the total biodiesel production. Since the soaring petroleum price has made oleochemicals, especially biodiesel production, increasingly attractive,15 glycerol production has rapidly moved into oversupply resulting in a glut on the market.16 Cheap glycerol, the price of which has fallen to about 10 cents per pound, is widely available on the EU and North American market.17 Although there is extensive utilization of high purity glycerol (>99.0%) in the food, cosmetic and pharmaceutical industries, it is technically difficult and costly to obtain pure glycerol during biodiesel production.18 It is, therefore, desirable to find a novel, economic use for cheap glycerol in order to mitigate the collapse in the price of glycerol and further defray the cost of biodiesel production.19,20 Little research has been done on the alternative use of glycerol in treating lignocellulosic biomass. ¨ uk ¨ The only example found was that of Kuc and Demirbas,21,22 whose research indicated that aqueous glycerol organosolv pulping can lead to high lignin removal from lignocellulosic biomass. Previous work by the authors not only confirmed this, but also found that atmospheric industrial glycerol autocatalytic organosolv pretreatment enabled lignocellulosic biomass to provide good enzymatic hydrolysis yield.23 It is, thus, of interest to further examine this form of enzymatic hydrolysis. In addition, coworkers24 – 26 and other researchers27 – 29 have reported that steam explosion pretreatment (SEP) is a cost-effective technique and has great potential in industrial applications. It could be very beneficial to perform fractional separation and then enhance the enzymatic hydrolysis of lignocellulosic biomass. Accordingly, this paper reports on an investigation and comparison of SEP and atmospheric aqueous glycerol autocatalytic organosolv pretreatment (AAGAOP) in terms of their effectiveness for the enhanced enzymatic hydrolysis of lignocellulosic biomass. Using scanning electron microscope (SEM) and Fourier transform infrared (FT-IR) analysis, good evidence of physical and chemical structural changes in pretreated lignocellulosic biomass were found to explain the enhancement mechanisms.

MATERIALS AND METHODS Materials Air-dried wheat straw was collected in Henan Province, China, and cut manually into pieces approximately 20 mm in length, dried to constant weight at 60 ◦ C and then stored as ‘original’ wheat straw in a sealed polyethylene plastic container. The average main components (% w/w) of the original wheat straw were as follows: 41.3% cellulose, 31.7% hemicellulose and 17.3% lignin. 708

Industrial glycerol of commercial grade (95% purity), was purchased from Hengshui Jinghua Chemical Plant, Hebei Province, China, and was diluted to a concentration of 70% (w/w) for use in experiments. Recycled industrial glycerol (RIG) liquor was collected from an industrial glycerol pretreatment process and vacuum concentrated to 70% for use. The AAGAOP process Previous work on AAGAOP established the optimum experimental conditions as 220 ◦ C for 3 h, with a liquid–solid ratio 200/10. Based on this, for a typical run, 10 g of dry wheat straw was suspended in 200 g of 70% aqueous glycerol or RIG liquor. The experiment was performed under reflux and heating with an electric heater, with temperature indicated by a thermometer. The temperature was kept at 220 ◦ C for 3 h and then allowed to cool. When the slurry had cooled to 120–130 ◦ C, it was supplemented slowly with 200 mL of recycled industrial glycerol liquor (approximately 40% consistency) and stirred vigorously to disintegrate the fiber. After thorough disintegration, the insoluble solid fiber fraction was separated by filtration through a G3 glass filter and washed twice with 400 mL recycled industrial glycerol liquor. After thorough washing with tap water, the insoluble solid fiber (‘pretreated’ wheat straw) was divided into two parts. One part was dried at 60 ◦ C to constant weight, to determine the pretreatment yields and main components, and the other part was conserved in a sealed bag at 5 ◦ C, the fiber fraction to be used for further enzymatic hydrolysis. The filtrate was treated for recycling. After precipitation overnight at room temperature, the spent liquor was centrifuged at 4000 rpm for 30 min. The supernatant was used directly as recycling liquor for washing, i.e. recycled industrial glycerol liquor (approximately 40% consistency). When used for cooking in the experiment, it was vacuum concentrated to 70% (w/w) using a rotary evaporator (RE-52A, Shanghai Yarong Biochemical Instrument Company, China). The SEP process Samples of 200 g wheat straw were cut into pieces approximately 10 cm in length and impregnated with 200 mL purified water overnight. Steam explosion of the impregnated wheat straw was performed using saturated steam in a 4.5 L batch reactor (Weihai Automatic Control Reactor Ltd., China), under optimal pretreatment conditions (1.5 atm, 198 ◦ C × 5 min, R0 = 3893) established previously.25,26 After each run, the steam exploded wheat straw was collected and then washed three times, each time with 1.5 L tap water. The washed steam exploded wheat straw was divided into two parts. One part was dried to determine the pretreatment yields and main components, and the other was conserved in a sealed J Chem Technol Biotechnol 83:707–714 (2008) DOI: 10.1002/jctb

Comparison of glycerol and steam explosion pretreatments of wheat straw

Enzymatic hydrolysis of the resulting fiber fractions Just as in earlier reports,23 a mixture of P. decumbens cellulase and β-glucosidase, supplied by Ningxia Cellulase Preparation Plant, China, was used to detect enzymatic convertibility of the fiber fraction to reducing sugar. Filter paper activity (FPA) of cellulase and β-glucosidase activity were determined as 110 FPU mL−1 and 42 IU mL−1 , according to the methods of Ghose30 and Kubicek,31 respectively. Each individual sample (approximately 0.5 g dry weight) of wet or dried solid fiber fraction from the pretreatment process was suspended quickly in 0.2 mol L−1 , pH 4.8 acetate buffer in a 100 mL flask, and diluted to 25.5 g slurry, which was achieved with approximately 2% solids (w/w) to reduce end-product inhibition. The slurry was then supplemented with cellulase enzymes of 44 FPU g−1 dry matter and β-glucosidase of 16.8 IU g−1 dry matter.23 Enzymatic hydrolysis was performed at 180 rpm in a rotary shaker at 50 ◦ C. Analysis The resulting insoluble solid fiber fraction was dried at 60 ◦ C to constant weight for dried weights determination. The main components (cellulose, hemicellulose and lignin) of the original wheat straw and resulting fibers were analyzed by a modified sequential gravimetric method.32,33 The pretreatment yields and components were designated, respectively, as follows: pretreatment yield (%) = 100 (g in insoluble solid fiber fraction) (g in wheat straw)−1 ; component yield (%) = 100 (g in component of insoluble solid fiber fraction) (g in component of wheat straw)−1 . All experiments were performed in duplicate under the same conditions and average values are reported. The standard deviations were less than 4%. The total reducing sugar was determined by the method of Miller34 and calculated as follows: enzymatic hydrolysis yield (%) = 0.9 (g in reducing sugar) 100 (g in carbohydrates)−1 . Each sample measurement was performed in duplicate, with the average value reported. The standard deviation was less than 3.2%. FT–IR spectra of the samples were obtained using a Fourier transform infrared spectrophotometer (Perkin-Elmer System 2000, USA) and KBr disc containing about 1% finely ground samples. Microgaphs were taken using a field emission SEM (JSM-6700F, JEOL, Japan) after the samples were sputtered with a thick layer of gold, spread uniformly from all sides and at two different angles. RESULT AND DISCUSSION Main components of wheat straw pretreated by the two methods Figure 1 illustrates the yields and main chemical components for wheat straw pretreated by the two J Chem Technol Biotechnol 83:707–714 (2008) DOI: 10.1002/jctb

methods. AAGAOP was carried out at 220 ◦ C for 3 h using (1) 70% industrial glycerol (IG) and (2) recycled industrial glycerol liquor (RIG); steam explosion (SE) pretreatment was at conditions 1.5 atm × 5 min (R0 = 3893). Compared with AAGAOP using IG and RIG liquor, SEP gave higher hemicellulose removal from the original wheat straw, reaching ∼80%, which was ∼12% and ∼20%, respectively, higher than with the former two. However, cellulose yield with SEP was low, only 93%, revealing non-negligible cellulose degradation. Meantime, SEP resulted in only 10% lignin removal, which was far lower than ∼64% obtained with IG and ∼57% with RIG. These results indicate that SEP could disrupt the compact structure of lignocellulosic biomass and remove some components, especially hemicellulose. AAGAOP using either IG or RIG gave strong cellulose retention while removing more lignin and hemicellulose, compared with SEP. Enzymatic hydrolysis of fiber with different pretreatment methods The wet insoluble solid fiber fraction from wheat straw pretreated using IG, RIG and SE, was used directly as a substrate for enzymatic hydrolysis. Enzymatic hydrolysis of the wet (approximately 0.5g dry weight) and its correspondingly oven-dried fiber was carried out at 50 ◦ C, and 180 rpm in 0.2 mol L−1 acetate buffer, pH 4.8, with enzyme additions of 44 FPU g−1 and 16.8 IU g−1 dry matter. The sample was withdrawn periodically to determine reducing sugar levels.23 The profile of enzymatic hydrolysis of pretreated wheat straw is shown in Fig. 2. The wet fiber of wheat straw pretreated using SE, achieved an enzymatic hydrolysis yield of ∼69% after 72 h. The enzymatic hydrolysis yields with IG and RIG were much higher, up to ∼91% and ∼87%, respectively. This indicates that besides IG liquor, RIG was also suitable as a

100 Yield of pretreatment and components, % the original

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Figure 2. Enzymatic hydrolysis of AAGAOP and SEP pretreated wheat straw.

cooking solvent to enhance enzymatic hydrolysis of lignocellulosic biomass. Although SE is an economical pretreatment method, and has attracted much interest, it led to only a limited increase in enzymatic hydrolysis yield. This can be explained partly by its chemical composition (shown in Fig. 1), especially the residual lignin (high, up to ∼90% of the original content), which is a key limiting factor in enzymatic hydrolysis.1 On the other hand, the data suggest that the AAGAOP technique, using IG and RIG, performed better than SEP in enhancing enzymatic hydrolysis of lignocellulosic biomass. A similar study by Pan et al.35 also confirmed that aqueous ethanol organosolv pretreatments improve enzymatic hydrolysis of wood fiber more than an SE method, even if the latter leads to lower residual lignin content. Additionally, for steam-exploded wheat straw, the enzymatic hydrolysis yield of wet substrate increased by over 10% more than that of dried substrate. This argues again that wet fiber with rich moisture taken directly as the substrate is preferable for enzymatic hydrolysis. This argument is supported by the recent finding that structural modification of residual lignin and carbohydrates during oven drying, forming lignin recondensation and/or lignin–carbohydrates complex (LCC), inhibited enzymatic hydrolysis.36 SEM observations of pretreated fiber fraction The above results on enzymatic hydrolysis of pretreated fiber by AAGAOP and SEP were such that it was of interest to further investigate the mechanisms occurring. For this purpose, SEM micrographs were taken of the original wheat straw and the fiber resulting from AAGAOP and SEP, as shown in Fig. 3. Figure 3(d) and 3(e) are higher magnification versions of Fig. 2(b) and (c) showing the long split fasciculus fully outside, indicating that AAGAOP and SEP both collapse the compact physical–chemical structure of lignocelluosic biomass. On the other hand, the long fibrils disrupted by AAGAOP (Fig. 3(c) 710

and 3(e)) were more severely damaged, non-compact and finer than those for SEP (Fig. 3(b) and 3(d)), which led to the former having greater roughness and surface area than the latter. The small average fiber size, and increased roughness and surface area rendered the fasciculi more susceptible to enzyme access and attack, and hence higher enzymatic hydrolysis yield.37 Thus, observation of the outer structure of pretreated fiber fraction enables an explanation of why the enzymatic hydrolysis obtained with AAGAOP was much higher than that obtained with SEP. FT-IR spectra of wheat straw fiber before and after pretreatment In order to explore in detail the mechanisms involved in the two pretreatment methods, an investigation of the chemical structural changes occurring in pretreated lignocellulosic biomass was carried out using FT-IR spectroscopy. Figure 4 shows the FTIR spectrum of a fiber fraction from the original wheat straw, from IG pretreated wheat straw, and from SE wheat straw. The band at 1648 cm−1 is indicative of the bending mode of the absorbed water. An intensive and sharp band at 1049 cm−1 in spectra A, B and C is attributed to the C–O–C stretching typical of glucan and xylan. Peaks at 1425 cm−1 and 1382 cm−1 are due to C–H and OH bending, and CH2 and OH bending, respectively.38 In the anomeric region (950–700 cm−1 ), a small sharp band at 899 cm−1 in spectra A, B and C is characteristic of βglycosidic linkages. This demonstrates the presence of predominant β-glycosidic linkages between the sugar units in the fiber fraction.39 Peaks at 1159 cm−1 and 1107 cm−1 , which increase in spectra B and C, arise from C–O anti-symmetric bridge stretching and C–OH skeletal vibration, respectively.40 In contrast with spectrum A, the ester bonds (C=O) signal at 1735 cm−1 is much weaker in spectrum B and is absent in spectrum C. The data indicate that the important ester linkages were dissociated between ferulic acid or p-coumaric acid or (p-) hydroxycinnamic acids and lignins in the pretreated fiber, which is consistent with the report by Chen and Liu.26 Moreover, the AAGAOP was more competitive for SEP in disrupting the key ester linkages between lignin and carbohydrates. On the other hand, there is no obvious change of the bond at 1321 cm−1 (syringyl units) in spectra B and C. However, the –C–O–stretching band (guaiacyl units) at 1249 cm−1 is decreased significantly in spectrum B and almost disappears in spectrum C. The information indicates that the relative content of syringyl lignin units in the steam-exploded and glycerol pretreated fibers have no significant changes compared with the original wheat straw. But their guaiacyl lignin units are reduced greatly from the steam-exploded to glycerol-pretreated.41 It is, therefore, inferred that the two pretreatment methods had active roles in J Chem Technol Biotechnol 83:707–714 (2008) DOI: 10.1002/jctb

Comparison of glycerol and steam explosion pretreatments of wheat straw

(a)

(b)

(c)

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Figure 3. SEM images of wheat straw fiber before and after pretreatment: (a) original fiber (1000×); (b) fiber from SEP (300×); (c) fiber from AAGAOP (300×); (d) fiber from SEP (1000×); (e):fiber from AAGAOP (1000×).

J Chem Technol Biotechnol 83:707–714 (2008) DOI: 10.1002/jctb

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Figure 4. FT-IR spectrum of dried fiber fraction (A) from the original wheat straw; (B) from SEP; and (C) from AAGAOP.

guaiacyl lignin removal and relatively small effects on the syringyl lignin. Also, the AAGAOP was more effective than SEP in disrupting the guaiacyl lignin units of the residual lignin. Despite no obvious change of peak intensity at 1321 cm−1 (syringyl lignin) in pretreated fiber, AAGAOP and SEP both removed syringyl lignin. Moreover, the former led to more removal than the latter, as indicated by the different pretreatment yields given by the two pretreatment methods. Interestingly, peaks at 1510 cm−1 and 1457 cm−1 , representing associated lignin components and its aromatic ring stretch, respectively, intensified in spectrum B and fell into a shoulder on spectrum C. This indicates that large amounts of residual lignin still bond to carbohydrates in the steam-exploded fiber, while only a little remained in the AAGAOP fiber. This corresponds with the residual lignin content of pretreated fiber (Fig. 1). The result also confirms that AAGAOP delignified more effectively than SEP. All results indicate that both pretreatment methods disrupted important ester linkages between lignin and carbohydrates in the lignocellulosic fiber fraction. They gave priority to dissociating the guaiacyl units of residual lignin and played a relatively small role in removal of syringyl units. Moreover, AAGAOP, as a novel effective pretreatment technique, appeared more competitive than SEP.

DISCUSSION This study compared AAGAOP and SEP by examining chemical composition, enzymatic digestion and the physical–chemical structure of pretreated wheat straw. Results indicate that both pretreatment methods were capable of preserving large amounts of glucan (≥90% of the original cellulose), removing high levels of xylan (≥70% of the original hemicellulose) and enhancing enzymatic digestion (≥65% of the theoretical maximum). These results were achieved by autocatalysis, where acetic acid and other organic acids are liberated from hemicellulose and act in a catalytic role. 712

This point was indicated by pH degradation (data not shown) occurring frequently in the AAGAOP and SE process. Further, changes of peak intensity at 1735 cm−1 and 1249 cm−1 (Fig. 4), indicating acetyl and uronic ester groups or the ester bonds of carboxylic groups of the ferulic or p-coumaric acid, also confirmed this. Regarding the economic benefits of autohydrolysis, these would be no corrosion, low capital and operating costs, and less cellulose degradation under normal operating conditions.42 When assessing the effect of SE on lignocellulosic biomass, such factors as pretreatment temperature and retention time are generally taken into consideration. The conditions 1.5 atm × 5 min (R0 = 3893) adopted in this work was less severe than that used in some earlier studies.6,27 Although the mild pretreatment led to low delignification (∼10% of the original lignin) and a limited increase in enzymatic hydrolysis (∼69%), it could reduce the degradation of cellulose and the in situ production of inhibiting compounds, with low energy consumption, compared with more severe steam explosion conditions.28,29 Regarding AAGAOP, this gave high cellulose recovery (≥95%) and enzymatic hydrolysis yield (∼90%) based on simultaneous good delignification (≥65%) and hemicellulose removal (≥70%).6,27 Some further specific advantages of the AAGAOP technique were as follows: first, the glycerol liquor used in the AAGAOP process can be completely recycled and reused to treat lignocellulosic biomass to enhance enzymatic hydrolysis, thus reusing process waters and therefore meeting environmental requirements. Second, the cooking process of AAGAOP involves lignocellulosic biomass in an organic solvent–water media, so it has low environmental impact and less energy consumption. Third, glycerol, a high-boilingpoint solvent used in the process, is operated at pressure levels similar to or even lower than in traditional Kraft processes.12,13 Finally, owing to the highly polar polyalcohol structure, glycerol easily penetrates into the fiber tissue, providing an effective reaction medium for delignification of lignocellulosic biomass.21,22 CONCLUSIONS AAGAOP and low-severity SEP were both suitable as autocatalytic pretreatments of lignocellulosic biomass to enhance enzymatic hydrolysis by removing chemical compositional obstacles and by modifying the physical structure. Based on high cellulose retention, AAGAOP resulted in simultaneous good lignin and hemicelluloses removal, while the main effect of SEP was only on hemicellulose removal. Regarding delignification, the two methods gave priority to dissociating the guaiacyl units of residual lignin and had a relatively small role in the removal of syringyl units. The AAGAOP technique is considered more competitive than SEP in enhancing the recovery and enzymatic digestibility of cellulose. J Chem Technol Biotechnol 83:707–714 (2008) DOI: 10.1002/jctb

Comparison of glycerol and steam explosion pretreatments of wheat straw

ACKNOWLEDGMENTS This work was financially supported by National Basic Research Program of China (973 Project, No. 2004CB719700) and National Key Project of Scientific and Technical Supporting Programs Funded by the Ministry of Science & Technology of China during the 11th Five-year Plan Period (No. 2006BAD07A07).

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J Chem Technol Biotechnol 83:707–714 (2008) DOI: 10.1002/jctb