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Effects of Crystallinity on Dilute Acid Hydrolysis of Cellulose by Cellulose Ball-Milling Study Haibo Zhao, Ja Hun Kwak, Yong Wang, James A. Franz, John M. White, and Johnathan E. Holladay* Pacific Northwest National Laboratory, Institute for Interfacial Catalysis, P.O. Box 999, Richland, Washington 99352 ReceiVed September 27, 2005. ReVised Manuscript ReceiVed NoVember 23, 2005
The dilute acid (0.05 M H2SO4) hydrolysis at 175 °C of samples comprised of varying fractions of crystalline (R-form) and amorphous cellulose was studied. The amorphous content, based on XRD and CP/MAS NMR, and the product (glucose) yield, based on HPLC, increased by as much as a factor of 3 upon ball milling. These results are interpreted in terms of a model involving mechanical disruption of crystallinity by breaking hydrogen bonds in R-cellulose, opening up the structure, and making more β-1,4 glycosidic bonds readily accessible to the dilute acid. However, in parallel with hydrolysis to form liquid-phase products, there are reactions of amorphous cellulose that form solid degradation products.
1. Introduction Cellulose is the most abundant renewable carbon source available and can potentially meet our future energy needs if cellulose can be efficiently converted to monomeric sugars. A single cellulose molecule is an unbranched chain of glucose units linked head to tail. Compared to starch, the hydrolysis of native cellulose is much more difficult. Cellulose forms two crystalline forms, IR and Iβ, in statistically variable proportions.1 In crystalline cellulose, each cellulose chain approximates to a flat ribbon, with alternate glucose units facing in opposite directions.2 All the cellulose chains lie parallel, hydrogen-bonded edge to edge. The sheets of chains so formed are stacked on top of one another with a stagger, along the microfibril, which differs between the IR and Iβ forms. These structures are well characterized by synchrotron X-ray and neutron fiber diffraction.3,4 The weak C-H‚‚‚O hydrogen bonding is thought to be the force that holds sheets of chains together in stacks.2,3 R-Cellulose was used in our study because it is more abundant in nature than β-cellulose. The crystal structure and hydrogen bonding in cellulose greatly limit the access to β-1,4-glycosidic bonds by reactants and catalysts. Water is excluded almost completely from the crystalline regions in cellulose.5-7 This limitation makes cellulose hydrolysis much slower than that of starch. The amorphous part of cellulose is more readily accessible by water and other reactants.8 Molecular dynamics simulations * Corresponding author. Tel: 509-375-2025. Fax: 509-372-4732. Email:
[email protected]. (1) Atalla, R. H.; Vanderhart, D. L. Science 1984, 223, 283. (2) Jarvis, M. Nature 2003, 426, 611. (3) Nishiyama, Y.; Sugiyama, J.; Chanzy, H.; Langan, P. J. Am. Chem. Soc. 2003, 125, 14300. (4) Nishiyama, Y.; Langan, P.; Chanzy, H. J. Am. Chem. Soc. 2002, 124, 9074. (5) Child, T. F. Polymer 1972, 13, 259. (6) Hatakeyama, T.; Ikeda, Y.; Hatakeyama, H. Makromol. Chem. 1987, 188, 1875. (7) Nakamura, K.; Hatakeyama, T.; Hatakeyama, H. Text. Res. J. 1983, 11, 682. (8) Vittadini, E.; Dickinson, L. C.; Chinachoti, P. C. Carbohydr. Polym. 2001, 46, 49.
by Mazeau and Heux show that the total number of hydrogen bonds per repeat units is eight in the IR crystalline form and is only 5.3 in the amorphous form.9 They also show that the crystalline form of cellulose has much larger cohesive energy density than noncrystalline forms.9 This suggests that noncrystalline forms of cellulose will be more reactive. The acid-catalyzed hydrolysis of crystalline cellulose is a heterogeneous reaction, and models are well described in the literature.10-13 Even with continued research and development, breakthroughs are needed to raise the glucose yields above 65%. Two factors limiting glucose yield are glucose degradation14 and cellulose modification15 under various reaction conditions since temperatures higher than 200 °C are often used in traditional dilute acid cellulose hydrolysis processes. To reduce glucose degradation and cellulose modification, less harsh reaction conditions should be used in cellulose hydrolysis, but this will greatly decrease hydrolysis rate. Pretreating cellulose to increase the noncrystalline fraction will increase the number of accessible β-1,4-glycosidic bonds and likely reduce reaction temperature and time. Mild reaction conditions could greatly reduce glucose and cellulose degradation during reactions. The reprecipitated cellulose formed from that dissolved in 65% concentrated sulfuric acid solution can be hydrolyzed much faster than its original form because it is less crystalline.16 Increasing the noncrystalline cellulose fraction may also accelerate cellulose enzyme hydrolysis processes due to an increase of accessible β-1,4-glycosidic bonds. In this paper, we physically decreased cellulose crystallinity by the ball milling of R-cellulose. Although this pretreatment (9) Mazeau, K.; Heux, L. J. Phys. Chem. B 2003, 107, 2394. (10) Mok, W. S.; Antal, M. J. Ind. Eng. Chem. Res. 1992, 31, 94. (11) Torget, R. W.; Kim, J. S.; Lee, Y. Y. Ind. Eng. Chem. Res. 2000, 39, 2817. (12) Xiang, Q.; Kim, J. S.; Lee, Y. Y. Appl. Biochem. Biotechnol. 2003, 105, 337. (13) Saeman, J. F. Ind. Eng. Chem. 1945, 37, 43. (14) Corner, A. H.; Wood, B. F.; Hill, C. G.; Harris, J. F. J. Wood Chem. Technol. 1985, 5, 461. (15) Bouchard, J.; Abatzoglou, N.; Chornet, E.; Overend, R. P. Wood Sci. Technol. 1989, 23, 343. (16) Xiang, Q.; Lee, Y. Y.; Pettersson, P. O.; Torget, R. W. Appl. Biochem. Biotechnol. 2003, 105-108, 505.
10.1021/ef050319a CCC: $33.50 © 2006 American Chemical Society Published on Web 12/23/2005
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is not viable in its practical nature because of the high energy input, the purpose of our work was to correlate hydrolysis reactivity with decreasing cellulose crystallinity. Cellulose structures after ball milling were characterized by X-ray diffraction (XRD) and cross polarization/magic-angle spinning 13C solid-state nuclear magnetic resonance (CP/MAS NMR). We examined the influence of crystallinity on hydrolysis in dilute sulfuric acid at a relatively low reaction temperature. 2. Materials and Methods 2.1. R-Cellulose. R-Cellulose was purchased directly from Sigma-Aldrich (product no. C6663). Ball-milling experiments were performed on an US Stoneware ball-mill machine. A ZrO2 ball (mass of 1 kg and diameter of 1 cm) and 30 g of R-cellulose were loaded into a polypropylene bottle (500 mL). Spinning speed was set at 60 rpm. Five grams of cellulose samples were taken out at a desired time (1, 2, and 6 days) and used in our experiments. 2.2. X-ray Diffraction Method (XRD). XRD measurements were performed on a Philips PW3040/00 X′Pert MPD system. The diffracted intensity of Cu KR radiation (wavelength of 0.1542 nm, under conditions of 50 kV and 40 mA) was measured in a 2θ range between 10° and 50°. 2.3. CP/MAS 13C Solid-State NMR. Cross polarization/magicangle spinning (CP/MAS) 13C solid-state NMR experiments were performed on a Chemagnetics CMX100 spectrometer operating under a static field strength of 2.3 T (100 MHZ 1H) at 25 °C. The contact time for CP was 1 ms with a proton pulse of 5.5 µs and decoupling power of 45 kHz. The MAS speed was 3 kHz. The delay time after the acquisition of the FID signal was 2 s. The chemical shifts were calibrated by using the hexamethylbenzene methyl resonance at 17.3 ppm. The cellulose crystallinity index (ICr), was determined from the peak areas assigned to C4 crystalline (86-92 ppm) and C4 noncrystalline (79-86 ppm) material. The cellulose crystallinity index is defined as ICr ) A86-92 ppm/(A79-86 ppm + A86-92 ppm) × 100% by deconvolution using a Lorenzian line shape, as discussed by Liitia¨ et al.17 2.4. Reaction Test. Reaction tests were performed using sealed tubular reactors. The reactors (∼15 cm3 internal volume) were constructed of Hastelloy C-276 tubing (0.5-in. OD × 6-in. length) capped with Swagelok end fittings. In our experiments, 0.8 g of cellulose and 8.0 g of 0.05 M sulfuric acid solution were loaded into a reactor. Reactors were heated to 175 °C in a fluidized sand bath, held for a selected reaction time, and taken out and quickly quenched in cold water bath. The liquids and solid residues were separated by filtration. HPLC was used to separate and quantify liquid products. Solid residues were washed with deionized water, dried at 105 °C overnight in a vacuum drier, and analyzed by CP/ MAS 13C solid-state NMR. We defined the yield so that for a 100% glucose yield all the initial cellulose is converted to glucose. 2.5. HPLC Analysis of Liquid Products. Liquid products were analyzed by HPLC using a Bio-Rad Aminex HPX-97H column. A refractive index detector was used in our analysis.
3. Results and Discussions 3.1. Ball-Milling Effects on Cellulose Crystallinity. Figure 1 shows cellulose XRD patterns for samples unmilled and ballmilled for 1, 2, or 6 days. The strongest peak, at 2θ ) 22.6°, originates from the cellulose crystalline plane 002.18,19 The intensities of peaks from other crystal planes also decrease as ball-milling time increases (Figure 1). Clearly, mechanical ball milling reduces the long-range order. In addition, we estimated (17) Liitia¨, T.; Maunu, S. L.; Hortling, B.; Tamminen, T.; Pekkala, O.; Varhimo, A. Cellulose 2003, 10, 307. (18) Park, C. H.; Kang, Y. K.; Im, S. S. J. Appl. Polym. Sci. 2004, 94, 248. (19) Newman, R. H. Solid State Nucl. Magn. Reson. 1999, 15, 21.
Figure 1. XRD patterns of as-received R-cellulose and ball milling for 2, 4, and 6 days as indicated.
the lateral dimensions of our cellulose samples to be 7.3 nm using the Scherrer equation:20
L ) Kλ/(β cos θ) where the X-ray wavelength is λ, the angle between incident and diffracted rays is 2θ, and K is a constant with a value of 0.94. Figure 2 shows the cellulose CP/MAS NMR spectra with and without ball milling. The milling procedure is expected to affect the crystallinity of the cellulose, which is confirmed by our XRD data. Consistent with this expectation, the peak ratios of C4(86-92 ppm)/C4(79-86 ppm) and C6(63-67 ppm)/C6(56-63 ppm) also decrease with increased ball-milling time, which clearly suggests that more disordered cellulose is produced in the ball-milling process. Even though CP/MAS NMR has been widely used to study cellulose structures, the underlying reasons for the shape of the C4 resonance (79-92 ppm) remain controversial. Two peaks are detected in most cellulose samples. The assignment of the peak between 86 and 92 ppm to the crystalline region is well accepted. The peak between 79 and 86 ppm is usually assigned either to the crystal surface or the disordered component in cellulose;21 however, it may also comprise a mixture of the two, since native cellulose always includes some noncrystalline regions.1,22 Recently, Kono et al. prepared IR- and Iβ-rich cellulose samples with no noncrystalline region.23 Their CP/ MAS NMR spectra show almost no signal between 79 and 86 ppm, which strongly suggests that this peak must originate from noncrystalline regions. The crystallinity index I which is the ratio between the C4 area of crystalline cellulose and the total C4 peak areas, can be used to define the crystallinity of cellulose.17,24 The cellulose crystallinity decreased from 0.77 for the as-received material to 0.52 after ball milling for 6 days. (20) Murdock, C. C. Phys. ReV. 1930, 35, 8. (21) Atalla, R. H.; Vanderhart, D. L. Solid State Nucl. Magn. Reson. 1999, 15, 1. (22) Vanderhart, D. L.; Atalla, R. H. Macromolecules 1984, 17, 1465. (23) Kono, H.; Yunoki, S.; Shikana, T.; Fujiwara, M.; Erata, T.; Takai, M. J. Am. Chem. Soc. 2002, 124, 7506. (24) Lenholm, H.; Larsson, T.; Iversen, T. Carbohydr. Res. 1994, 261, 119.
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Figure 2. CP/MAS 13C solid-state NMR spectra of as-received R-cellulose and ball milling for 1, 2, and 6 days as indicated. The crystallinity index, Icr, is indicated on each curve and is calculated as ICr ) A86-92 ppm/(A79-86 ppm + A86-92 ppm) × 100%.
3.2. Reactivity of Ball-Milled Cellulose. To evaluate the reactivity of ball-milled cellulose, cellulose hydrolysis was carried out at low temperature (175 °C) with a dilute sulfuric acid (0.05 M) catalyst. Other experiments (not shown) suggest that when the as-received R-cellulose is used in the reaction, crystallinity and structure are not altered under these reaction conditions. Hydrolysis occurs to a small extent and produces hydroxymethylfurfuraldehyde (HMF) and levulinic acid, presumably via glucose formation followed by glucose degradation. Figure 3 shows the glucose yield at various reaction times from the hydrolysis of as-received and ball-milled cellulose samples. The glucose yield increases with ball-milling time and reaction time. The latter observation implies that the rate of glucose formation exceeds the rate of glucose degradation. Under various reaction conditions glucose (C6 molecule) can react to form HMF (C6 molecule), which can further react to form levulinic acid (C5 molecule). To better understand the cellulose hydrolysis process, we plotted total C5 and C6 product yields with reaction time in Figure 4. This combined number represents real hydrolysis yields by including glucose degradation products. Increasing cellulose ball-milling time from 0 to 6 days more than doubles the total C5 and C6 produced. The yield also increases with increasing reaction time, but the rate drops, particularly for reaction times exceeding 20 min. The decreasing rate may be due, in part, to changes in the cellulose since it darkened with increasing reaction time. Perhaps a small amount of cellulose degrades or glucose degradation products adsorb on the cellulose surface. Amounts of degradation products adsorbed on exposed surfaces could be sufficient to inhibit the hydrolysis rate without being detectable in CP/MAS NMR. The comparison of Figures 3 and 4 suggests that increasing ball-milling time increased the total conversion of cellulose, but it also increased the yield of glucose degradation products because glucose degradation will increase with increasing glucose concentration in the reactants.
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Figure 3. Glucose yields at different reaction times from cellulose ball-milled for different numbers of days.
Figure 4. Total C5 + C6 products yields at different reaction times from cellulose ball-milled for different numbers of days.
It has been reported that cellulose acid hydrolysis rates are limited in two general wayssintrinsic cleavage of glycosidic linkages and physical access to these linkages. When the cellulose that dissolved in 65% H2SO4 solution was precipitated, the so-called reprecipitated cellulose (amorphous) was hydrolyzed 100-fold faster than crystalline R-cellulose.16 The reprecipitated cellulose is claimed to be hydrolyzed at the same rate as corn starch under identical reaction conditions.16 Consistent
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with the ball-milling results, this is attributed to minimal intermolecular hydrogen bonding in the reprecipitated cellulose. Schwanninger and co-workers25 suggested that changes in the FTIR spectrum of cellulose after ball milling for 1 min were caused by the disruption of hydrogen bonds. However, our XRD and CP/MAS NMR experiments (not shown) exhibit no evidence for alteration of the cellulose structure after 1 h of ball milling. The FTIR spectra may be more sensitive than XRD and CP/MAS NMR to disruption of the weak C-H‚‚‚O hydrogen bonding that holds sheets of chains together in a stack. Ball milling for longer times delivers mechanical stress sufficient to be detected by XRD and CP/MAS NMR. Efficiently disrupting hydrogen bonding, making many more β-1,4-glycosidic bonds accessible to reactants and catalysts, is critical to increasing the cellulose hydrolysis rate. Sasaki and co-workers26 found that cellulose is rapidly dissolved and depolymerized in supercritical water at 300-320 °C with no acid. They suggested that supercritical water completely disrupts the intramolecular and intermolecular hydrogen-bond linkages. Dissolving the cellulose eliminates one of the restrictions of the cellulose physical state on the hydrolysis reaction, and the reaction changes from heterogeneous to homogeneous. Concentrated acids can also dissolve cellulose and eliminate physical-state effect on the hydrolysis reaction, but cost and environmental concerns make these processes unattractive or impractical. Safe, effective solvents or other methods for breaking hydrogen bonds in cellulose are needed to make cellulose hydrolysis more competitive. 3.3. Cellulose Crystallinity Change in the Hydrolysis. Cellulose cystallinity and structure were not altered under our reaction conditions when R-cellulose was used in the reactions although noncrystalline regions exist in R-cellulose. The possible reason is that these noncrystalline regions are surrounded by crystalline regions. Figure 5 shows cellulose CP/MAS NMR spectra both before and after reaction for a ball-milled (6 days) sample. The cellulose crystallinity increases with increasing reaction times. This is expected because the amorphous fraction will be hydrolyzed selectively leaving crystalline cellulose intact. These results are consistent with the idea that the noncrystalline material formed by ball milling is located preferentially at the surface of crystalline material where it can be accessed easily by acid and water. 3.4. Stability of Ball-Milled Cellulose under Hydrolysis Conditions. Figure 6 shows cellulose CP/MAS NMR spectra before and after hydrolysis. The curves are for R-cellulose, and the samples are coded as follows: A: as-received; B: asreceived and hydrolyzed 40 min; C: ball-milled 2 days and 40 min reaction; D: ball-milled 6 days and 40 min reaction. The CP/MAS NMR spectra of A and B are indistinguishable even though, visually, the color changed from white to yellow during hydrolysis. Evidently, the extent of hydrolysis was not detectable by CP/MAS NMR. Sample C (ball-milled 2 days) gave distinguishing features in the CP/MAS NMR spectra of the remaining solid. Small peaks at 20 and 25 ppm are characteristic of methyl groups, and the broad features near 150 ppm are attributable to CdC double bonds. The peak at 43 ppm remains unassigned. The CP/MAS NMR spectra indicate the cellulose degradation during hydrolysis increase as with ball-milling time. The intensities of peaks due to cellulose degradation increase, and new small peaks near 200 ppm indicate the formation of (25) Schwanninger, M.; Rodrigues, J. C.; Pereira, H.; Hinterstoisser, B. Vib. Spectrosc. 2004, 36, 23. (26) Sasaki, M.; Fang, Z.; Fukushima, Y.; Adschiri, T.; Arai, K. Ind. Eng. Chem. Res. 2000, 39, 2883.
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Figure 5. CP/MAS 13C solid-state NMR spectra before and after reaction (5 min, 10 min) for a ball-milled (6 days) sample.
Figure 6. Cellulose CP/MAS NMR spectra before and after hydrolysis. The curves are for R-cellulose, and the samples are coded as follows: A: as-received; B: as-received and hydrolyzed 40 min; C: ball-milled 2 days and 40 min reaction; D: ball-milled 6 days and 40 min reaction.
CdO groups. Consistent with these findings, the color of cellulose after hydrolysis becomes much darker with increasing ball-milling time. Mazeau and Heux used molecular dynamics simulation to calculate the cohesive energy density of cellulose.9 The cohesive energy density of IR cellulose is 46 kcal/mol larger than that of amorphous cellulose, which suggests that crystalline cellulose is much more stable than amorphous cellulose. Confirming that amorphous cellulose is not thermodynamically stable, early DSC
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studies27 also show that the transition from amorphous to crystalline structure is exothermic. Water evolution from dehydration is usually the first step for cellulose degradation,28 and this process can be catalyzed by acid. Dehydration involves OH groups, and free OH groups may react with lower activation energy than those that are hydrogen-bonded. Since amorphous cellulose has more free OH groups, this form will be more vulnerable to undesirable degradation. These findings suggest that less harsh conditions or shorter reaction times may have to be used to reduce degradation of amorphous cellulose during acid hydrolysis. 4. Conclusions Mechanical ball milling significantly increases the noncrystalline fraction in crystalline R-cellulose. The decreased crystallinity is evidenced by decreased crystalline peak intensity in XRD and a crystallinity index decrease in CP/MAS NMR. The crystallinity index of cellulose decreased from 0.773 to 0.523, and the cellulose hydrolysis rate increase more than doubled after R-cellulose was ball-milled for 6 days. The increased hydrolysis rate was attributed to disruption of hydrogen bonding and a concomitant increase in the number of β-1,4-glycosidic (27) Kimura, M.; Hatakeyama, T.; Nakano, J. J. Appl. Polym. Soc. 1974, 18, 3069. (28) Scheirs, J.; Camino, G.; Tumiatti, W. Eur. Polym. J. 2001, 37, 933.
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bonds accessible to the acid involved in hydrolysis. During hydrolysis, the crystallinity increases since the noncrystalline cellulose was preferentially hydrolyzed. As-received R-cellulose is very stable under our hydrolysis reaction conditions; after hydrolysis, CP/MAS NMR of the remaining solid exhibited nothing other than cellulose. The results differ for ball-milled samples; after hydrolysis for 40 min, CP/MAS NMR spectra of the remaining solid contained new features attributable to CH3, CdO and CdC groups. This could be due to increased numbers of free OH groups resulting from ball milling. The increased number of free OH groups increases the number of dehydration sites in cellulose, allowing for an increase of the average cellulose degradation rate. Acknowledgment. The authors thank Dr. James F. White, Dr. Charles H. F. Peden, and Dr. Thomas H. Peterson for helpful and informative discussions and thank Alan R. Cooper and Danielle S. Muzatko for initial HPLC analysis. This work was supported by the Laboratory Directed Research and Development Program at the Pacific Northwest National Laboratory (PNNL), a multiprogram national laboratory operated by Battelle for the U.S. Department of Energy under Contract DE-AC06-76RL01830. Part of the research described in this paper was performed at the Environmental Molecular Science Laboratory, a national scientific user facility located at PNNL. EF050319A
