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Decomposition of Cellulose in Near-Critical Water and Fermentability of the Products Tsuyoshi Sakaki,* Masao Shibata, Toshiharu Miki, and Hideharu Hirosue Kyushu National Industrial Research Institute, Tosu, Saga 841, Japan
Nobuyuki Hayashi Faculty of Agriculture, Saga University, Honjo, Saga 840, Japan Received August 7, 1995. Revised Manuscript Received January 24, 1996X
The noncatalytic decomposition characteristics of cellulose in near-critical water were examined by heating a sealed reactor in which the cellulose and water were charged in a salt bath kept at 305, 355, or 405 °C. Cellulose was rapidly decomposed to water solubles (WS), and the WS was further decomposed after the WS yield reached nearly 80%. The heating time giving the maximum WS yield was shortened to under 15 s by increasing the treatment temperature to over 355 °C. In the WS formation process, hydrolysis preferentially occurred, and the glucose yield reached 40% by the treatment for 15 s in the bath kept at 355 °C. On entering the second decomposition process, the WS was converted to gaseous products and methanol-soluble products, and charlike solid products were formed from the liquid phase. The hydrolysate of cellulose obtained in the WS formation process was subjected to a fermentation test, and the formed glucose was confirmed to be converted to ethanol.
Introduction In order to produce glucose, which is a highly significant substance for the production of alcohol and many other chemicals, from cellulosic materials at a high rate, many acid-catalyzed hydrolysis processes, concentrated1 and dilute2-4 acid ones, have been investigated. However, the acid-catalyzed hydrolyses performed under relatively mild temperature conditions (usually under 250 °C) have not achieved a sufficiently high reaction rate and moreover have a problem of corrosion of the reactor by the acid at the elevated temperature as well as processing of waste water. On the other hand, noncatalytic hydrothermal processes have also been studied.5-7 Bonn et al.6 achieved glucose conversions of up to 52% by hydrolyzing filter paper with pure water at 265 °C using a percolator reactor. Adschiri et al.8 increased the reaction rate by raising the treatment temperature. Cellulose was converted to water solubles with a glucose yield of 34% during the heat-up period in less than 15 s when the cellulose was heated with water at 400 °C. They reported that the overall reaction rate in near critical water was 1 or 2 orders of magnitude higher than those of the lower temperature acid catalytic processes. However, the decomposition charAbstract published in Advance ACS Abstracts, March 15, 1996. (1) Dunning, J. W.; Lathrop, E. C. Ind. Eng. Chem. 1945, 37, 2429. (2) Thompson, D.; Grethlein, H. E. Ind. Eng. Chem. Prod. Res. Dev. 1979, 18, 166-169. (3) Church, J.; Wooldridge, D. Ind. Eng. Chem. Prod. Res. Dev. 1981, 20, 371-378. (4) Mok,W. S‚L.; Antal, M. J.; Varhegyi, G. Ind. Eng. Chem. Res. 1992, 31, 94-100. (5) Bobleter, O.; Niesner, R.; Rohr, M. J. Appl. Polym. Sci. 1976, 20, 2083-2093. (6) Bonn, G.; Concin, R.; Bobleter, O. Wood Sci. Technol. 1983, 17, 195-202. (7) Bobleter, O. Prog. Polym. Sci. 1994, 19, 797-841. (8) Adschiri, T.; Hirose, S.; Malaluan, R.; Arai, K. J. Chem. Eng. Jpn. 1993, 26, 676-680. X
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acteristics of cellulose in near-critical water were not sufficiently clarified. Therefore, in this study, the decomposition characteristics of cellulose through noncatalytic and thermochemical treatment in near-critical water are examined. In addition, the fermentability of the decomposed cellulose is examined because substances which suppress the function of yeast may be generated during this thermochemical treatment. Experimental Section Apparatus and Procedure for Cellulose Decomposition. The cellulose used in this study is microcrystalline cellulose prepared from refined pulp for column chromatography (100-120 µm, Funakoshi Co.). The reactor used for the decomposition of cellulose is, as shown in Figure 1A, a tube reactor (SUS 316, 9.3 mm i.d. × 83 mm length, 6 mL) equipped with a thermocouple and a valve. Dried cellulose powder, 0.5 g, and distilled water, 3.0 g, were placed in the reactor. The air in the reactor was displaced with carbon dioxide as an inert gas, and then the reactor was sealed after carbon dioxide was charged at 0.1 MPa. Heating of the reactor was conducted in two steps using two salt baths as shown in Figure 1B. That is, the reactor was immersed in a preheater kept at 250 °C for 3 min and subsequently heated in a main heater for a predetermined time while shaking at ca. 250 times a minute. Then it was immediately cooled in a water bath to terminate the reaction. Figure 1C shows an example of the temperature profile of the reactor contents during this series of treatments. The reaction conditions are expressed by both the heating time in the main heater and the final temperature reached during the reaction period. Analytical Procedure. Figure 2 shows the analytical procedure after the treatments of preheating, reaction, and cooling. After the gas was exhausted, the contents in the reactor were thoroughly washed with hot water of ca. 40 °C and filtered using a No. 4 glass filter. The water in the filtrate was distilled off under vacuum, and the water solubles, WS, were obtained. The filter residue was washed with methanol
© 1996 American Chemical Society
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Figure 1. Apparatus and procedure for cellulose decomposition.
Figure 2. Analytical procedure. to examine the formation of water-insoluble fractions: methanol was found to be the best solvent for the decomposed cellulose products among methanol, acetone, benzene, and tetrahydrofuran by supplemental examinations. After thorough washing and filtration, methanol was removed from the filtrate and filter residue, and the methanol solubles, MS, and the methanol insolubles, MI, were obtained. WS, MS, and MI were weighed and each product yield was calculated based on the dry cellulose weight. The weight loss including gaseous products, G, was also obtained by subtracting the yields of WS, MS, and MI from 100. The component of WS was measured by HPLC equipped with two columns (SEC W12 + SEC W13, Yokogawa) and two detectors for ultraviolet (UV, 254 nm) and refractive index (RI)
in series. HPLC was operated at 40 °C with a 0.8 mL/min flow of a mixture of water and acetonitrile (70/30 by weight) as an eluate. The infrared spectra for MI were obtained with a Japan Spectroscopic IR-810 using the KBr pressed disk technique in which a mixture of 2 mg of sample and 200 mg of KBr was used to form the disk (1 mm thickness × 10 mm diameter). Fermentation Test. Several runs of cellulose decomposition treatments were carried out to collect the amount of hydrolysate required for a fermentation test. The heating time was 10 s and the average final temperature was 330 °C. After the separation of solid residue by filtration, 50 mL of hydrolysate solution was further supplemented with additional nutrients such as polypeptone, yeast extract, KH2PO4, and MgSO4 to final concentrations of 1.0, 0.5, 0.5, and 0.2%, respectively. The medium was adjusted to pH 6.0 with 0.1 N NaOH and autoclaved at 121 °C for 20 min in an Erlenmeyer flask. A loopful of yeast (Saccharomyces cerevisiae IFO 0216) from a stock culture (3.9% of potato dextrose agar, Nissui Chemical Co., Japan) was inoculated on the medium ((3 ( 0.6) × 108 cell/mL) and incubated stationary at 30 °C. As a control, 50 mL of 5% glucose solution was also subjected to the fermentation test under the same conditions as those for the hydrolysate solution. The amount of carbon dioxide formed during the fermentation was estimated by measuring the weight decrease of the sample. The sample before and after the fermentation was analyzed by HPLC (column, CHA S21 Yokogawa; detector, RI; temperature, 50 °C; eluate, water; flow rate, 1.0 mL/min) to confirm the formation of alcohol.
Results and Discussion Decomposition Characteristics. Figure 3 shows the yield of products as a function of heating time, where the reactor was heated in the main heater kept at 305 °C. Also shown with cross symbol is the final temperature of the reactant in the reactor. The temperature of the reactant rose rapidly from 250 to 305 °C. Cellulose, MI, was decomposed and the WS was formed with increased temperature and time. The WS yield increased to nearly 80% in 40 s. After that, the WS yield decreased and G and MS began to form instead. Figure 4 shows the results for the treatments conducted in the
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Figure 3. Product yields as a function of heating time; main heater, 305 °C.
Figure 4. Product yields as a function of heating time; main heater, 355 °C.
Figure 5. Product yields as a function of heating time; main heater, 405 °C.
main heater at 355 °C. The reaction rate was increased by raising the heating temperature. Significant reaction occurred during the heat-up period, and the WS yield reached the maximum when the temperature rose to 335 °C in 15 s. The cellulose residue once disappeared, and then the MI yield gradually increased together with the formation of G and MS. The heating time giving the maximum WS yield was further shortened to 13 s with no major change in decomposition behavior by raising the heating temperature to 405 °C as shown in Figure 5. Thus, it was confirmed that the decomposition rate of cellulose in near-critical water was extremely fast, and this noncatalytic decomposition process was found to be divided into the WS formation process and the second decomposition process. Analyses of Products. The components in WS were analyzed by HPLC equipped with UV and RI as detec-
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tors. A, B, C, and D in Figure 6 show the chromatograms for the WS obtained by the 12-, 14-, 17-, and 21-s heating in the main heater of 405 °C, respectively. That is, A and B are the results for the WS obtained in the WS formation process, and C and D are those for the second decomposition process. In the former process, glucose, which is a product of hydrolysis, was the primary component, and there were no clear peaks in the UV chromatograms excluding the peak of hydroxymethylfurfural (HMF), indicating that saccharification of cellulose through hydrolysis mainly progresses in the WS formation process. On the other hand, entering the second decomposition process, components having absorbance in UV began to form in the WS and many other peaks excluding sugars came out in the RI chromatograms, indicating that the WS is complicatedly modified by thermal decomposition during the second process. The change in the chemical structure of the solid residue, MI, with heating was also examined using IR. The spectra for the original cellulose and the MI obtained by the 14-, 15-, and 40-s heating in the main heater at 355 °C are shown as A, B, C, and D in Figure 7, respectively. The maximum WS yield was achieved by the 15-s heating in this case. Comparative analysis of IR in Figure 7 revealed no major changes in the chemical structure of the solid despite the large change in the MI yield during the WS formation process. The same result has been reported4 in the sufficiently dilute acid hydrolysis process of cellulose, although major structural changes have been observed9 to occur in the solid residue in the strong acid hydrolysis process. Acidcatalyzed parasitic reactions4 in the solid phase may have been avoided, as considered by Mok et al.,4 under the noncatalytic hydrothermal conditions. On the other hand, the chemical structure of MI formed in the second decomposition process was drastically different from that of the original cellulose. The peaks at 2900 and 900-1500 cm-1 disappeared and new peaks showing double bonds (CdO, CdC) appeared at 1700 and 1620 cm-1, indicating that this MI is no longer the carbohydrate type but a somewhat carbonized one.10 Because the cellulose residue almost disappears in the WS formation process (the minimum MI yield was 1.4% in 16 s in this case; Figure 4), the MI in the second decomposition process is concluded to have been formed from the liquid phase. Taking these results into account, the WS formation process is a process of saccharification of cellulose, where hydrolysis preferentially takes place and the formed oligomers dissolve in hot water to form the WS. The second decomposition process is a thermal degradation process of the WS, where the WS is decomposed to G and MS and charlike solid products are formed from the liquid phase. Fermentation of Decomposed Products. The amounts of glucose and cellobiose formed through the treatments conducted in the main heater of 305 °C are shown in Figure 8. The maximum yields of cellobiose and glucose reached 4 and 30% in 20 and 40 s, respectively. The maximum glucose yield was increased to 40% by raising the heating temperature to 355 °C as shown in Figure 9. However, a much higher heating temperature did not lead to the further enhancement of glucose formation as shown in Figure 10 where the (9) Bouchard, J.; Abatzoglou, N.; Chornet, E.; Overend, R. P. Wood Sci. Technol. 1989, 23, 343-355. (10) Tang, M. M.; Bacon, R. Carbon 1964, 2, 211-220.
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Figure 6. HPLC chromatograms for WS obtained under the conditions of 12 s heating time and 368 °C final temperature (A),14 s and 377 °C (B), 17 s and 381 °C (C), and 21 s and 389 °C (D). Peaks: c, cellobiose; g, glucose; h, HMF; ?, unknown component having absorbance in UV.
Figure 8. Glucose and cellobiose yields as a function of heating time; main heater, 305 °C.
Figure 7. IR spectra for original cellulose (A) and MI obtained under the conditions of 14 s heating time and 335 °C final temperature (B), 15 s and 337 °C (C), and 40 s and 355 °C (D).
treatments were conducted at 405 °C. The high glucose yield at a high rate despite the absence of a catalyst may be caused by the high ionization product11 of nearcritical water. In any treatment, the amount of cellobiose was under 5% and the maximum glucose yield appeared after the maximum cellobiose yield, implying that glucose is produced via an oligomeric intermediate as proposed by Abatzoglou et al.12 (11) Shaw, R. W.; Brill, T. B.; Clifford, A. A.; Eckert, C. A.; Franck, E. U. Chem. Eng. News 1991, 23, 26-39.
Figure 9. Glucose and cellobiose yields as a function of heating time; main heater, 355 °C.
Thus about 40% cellulose was found to be converted to glucose by adjusting the reaction conditions; however, HMF, known13 to be a substance inhibiting the activity (12) Abatzoglou, N.; Bouchard, J.; Chornet, E. Can. J. Chem. Eng. 1986, 64, 781-786.
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Figure 13. HPLC chromatograms for hydrolysate sample before fermentation (A) and after fermentation (B). Peaks: c, cellobiose; e, ethanol; g, glucose; x, components from the medium.
Figure 10. Glucose and cellobiose yields as a function of heating time; main heater, 405 °C.
Figure 11. Concentration of glucose, cellobiose and HMF in WS; main heater, 355 °C.
according to the fermentation reaction, C6H12O6 f 2C2H5OH + 2CO2. However, the fermentation rate of the hydrolysate solution was slower than that of the glucose solution, implying that the fermentation of the decomposed cellulose was inhibited due to the formation of inhibitors. The HPLC analysis of the hydrolysate sample before and after fermentation is shown in Figure 13. Quantitative analysis showed that 2.26 g of glucose was consumed from 2.57 g of glucose in the hydrolysate solution, 50 mL, and 1.18 g of ethanol was formed. Because 1.16 g of ethanol ought to be formed from 2.26 g of glucose according to the glucose fermentation reaction, essentially only glucose in the hydrolysate solution can be considered to have been converted to ethanol by S. cerevisiae IFO 0216. Thus the fermentation of the hydrolysate of cellulose obtained in the WS formation process was possible, although it has been reported14 that the fermentation hardly proceeds when the concentration of inhibitors is high. The level of byproducts such as HMF excluding oligomers is low in the heating time range where substantial amounts of glucose are formed (Figure 11); therefore, the fermentation of the products from the WS formation process should be profitable. However, the slow fermentation rate should be improved for industrialization, which will be investigated in our subsequent paper. Conclusions
Figure 12. Change in mole ratio of carbon dioxide to initial glucose with fermentation time.
of microorganisms for fermentation, was also formed as exemplified in Figure 11. Besides HMF, other inhibitors14 may also exist. Therefore, it is important to examine the fermentability of the product. The filtrate of the product obtained under the conditions of 10 s and 330 °C was subjected to a fermentation test. The change in mole ratio of carbon dioxide to the initial glucose with fermentation time is given in Figure 12. Also given is the result for the glucose solution as a control. Both the mole ratios increased toward 2 (13) Chung, I. S.; Lee, Y. Biotechnol. Bioeng. 1985, 27, 308-315. (14) Pfeifer, P. A.; Bonn, G.; Bobleter, O. Biotechnol Lett. 1984, 6, 541-546.
The noncatalytic decomposition characteristics of cellulose in near-critical water were examined by heating a sealed reactor in a salt bath kept at 305, 355, or 405 °C. The results are summarized as follows. As the reaction temperature and heating time increase, cellulose is decomposed to WS at a high rate and the WS is further decomposed after the WS yield reaches nearly 80%. The heating time giving the maximum WS yield is shortened to under 15 s by raising the bath temperature to over 355 °C. In the WS formation process, the hydrolysis preferentially progresses and the glucose yield from cellulose reaches 40% under a near-critical water condition. In the second decomposition process, the WS is decomposed to gaseous products and methanol-soluble products, and char-like solid products are formed from the liquid phase. The hydrolysate of cellulose obtained in the WS formation process can be subjected to fermentation, and the formed glucose is converted to ethanol. Acknowledgment. The authors thank Associate Professor T. Adschiri of Tohoku University for his helpful advice in arranging this paper. EF950160+
