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Ind. Eng. Chem. Res. 2004, 43, 2310-2314
Hydrothermal Production of Mono(galacturonic acid) and the Oligomers from Poly(galacturonic acid) with Water under Pressures Tetsuya Miyazawa and Toshitaka Funazukuri* Department of Applied Chemistry, Institute of Science and Engineering, Chuo University, 1-13-27 Kasuga, Bunkyo-ku, Tokyo 112-8551, Japan
A monosaccharide of galacturonic acid and its oligomers were produced by hydrolysis of poly(galacturonic acid)s with water in the absence of any additives in a semibatch flow reactor over the temperature range from 453 to 533 K at 10 MPa. A solid sample of poly(galacturonic acid) was fixed in the reactor, and the residence times of water in the reactor were estimated to be less than a few seconds. At 493 K and a heating time of 2 min, 79 wt % of the initial sample on the carbon weight basis was converted into water-soluble components, which consisted of monomer and oligomers having various degrees of polymerization (DP) and were enzymatically hydrolyzable into mono(galacturonic acid), the dimer, and the trimer. The maximum yields of monomer and oligomers with DP ) 2-10 were 11.4 and 22.1 wt %, respectively, on the carbon weight basis of the initial sample at 533 K, 10 MPa, and a heating time of 2 min. The yield of each product was correlated with the content of organic carbon in the eluted product solution. The formation of the further decomposed products such as furfural and gaseous products was insignificant under the hydrolysis conditions studied. Introduction Pectic substances, mainly consisting of R-(1-4)-linked D-galacturonic acid, are natural polysaccharides that are distributed in all higher plants and utilized as important materials in the food industry because of its gelling effect.1 Pectin, i.e., a kind of pectic substance, contains partly methyl-esterified galacturonic acid sequences as well as other neutral sugars such as rhamnose, galactose, etc.,2 although the composition is dependent on every ingredient.3,4 Poly(galacturonic acid) (PGA), whose molecular structure is shown in Figure 1 which is often referred to as pectic acid, is expressed as a de-esterified form of pectin. Pectic acid is water-insoluble, and one can obtain mono- and oligo(galacturonic acid) by hydrolysis.5,6 The oligomers play an important role as a defense mechanism in plants,7 and fermentation of galacturonic acid produces acetic acid and ethanol.8 The oligomers also have widely been employed as additives in the food industry, and they are commonly obtained by acid or enzymatic hydrolysis of pectin.5,6 To produce monomer and oligomers from hydrolysis of natural polymers, dilute acid, concentrated acid, and enzyme are employed as a hydrolysis medium. In addition to these media, hot compressed water,9 defined as water at a liquid state under pressures, has been recently paid attention as an attractive hydrolysis medium. The process was sometimes referred to as hydrothermolysis.10 Because of relatively high values of ionic product of [H+] and [OH-], e.g., approximately 10-11 mol2 kg-2 at 530 K and 10 MPa,11 almost no additive is required to hydrolyze polymers. In fact, some workers hydrolyzed lignocellulosic materials12-15 to produce monomers and their oligomers with water in the absence of any additives or at low concentrations of acid. However, the hydrolytic production of galacturonic acid and its oligomers with hot water under pressures has scarcely been reported in the literature. * To whom correspondence should be addressed. E-mail:
[email protected].
Figure 1. Molecular structure of PGA.
In this study we obtained mono(galacturonic acid) and its oligomers from the hydrolytic decomposition of PGA with hot water in the absence of any additives in a semibatch flow reactor having a relatively short residence time of water. We also investigated the effects of the reaction temperature on yields of products having various degree of polymerization (DP) values. Experimental Apparatus and Procedures A schematic diagram of the experimental apparatus is illustrated in Figure 2. The reactor made of stainless steel tubing (12.7 mm o.d., 8 cm length) was connected to a preheating column (1/8 in. stainless tubing of 2.17 mm i.d. and 2 m length). Two stainless steel tubings of 0.5 mm i.d., one (11 cm length) from the reactor outlet and the other to supply cold water to quench the eluted product solution, were joined with a T union. The line was further connected to a tubing (0.5 mm i.d., 44 cm length) equipped with a cooler jacket to quench the solution, followed by a back-pressure regulator (model 880-81; Jasco, Tokyo, Japan), which is capable of adjusting the pressure with fluctuation within (0.2 MPa with an electromagnetic, high-frequency openshut valve. The preheating column and reactor were
10.1021/ie0202672 CCC: $27.50 © 2004 American Chemical Society Published on Web 04/16/2004
Ind. Eng. Chem. Res., Vol. 43, No. 10, 2004 2311
Figure 4. TOC recovered vs reaction temperature. Figure 2. Schematic diagram of the experimental apparatus.
Figure 3. Time changes of temperatures measured in the reactor when the temperatures of the molten salt bath were 433, 493, and 533 K.
immersed in the molten salt bath whose temperature was maintained at the prescribed value within (2 K. At room temperature, a solid PGA sample of 0.5 g wrapped softly with quartz wool was placed in the reactor. A frit disk with 2 µm pore size was set at the exit of the reactor to fix the quartz wool. Soon after distilled and degassed water was supplied at a constantflow velocity of 10 mL min-1 by a high-performance liquid chromatography (HPLC) pump, the reactor was immersed in the molten salt bath, maintained at a prescribed temperature. The reaction time was counted from the moment the reactor had been immersed in the molten salt bath. The temperature of the reactor was found to quickly rise to 98% of the intended temperature within 1 min by measuring the temperature with a thermocouple placed in the reactor (see Figure 3). The distilled water at room temperature was also added to quench the solution, eluted from the reactor, at a flow rate of 10 mL min-1 and a pressure of 10 MPa by a HPLC pump (model PU-1580; Jasco). The product solution eluted from the back-pressure regulator was collected at certain intervals from 10 s to 3 min. Because the residence times of the fluid between the reactor inlet and the exit of the back-pressure regulator were about 15 s, obtained from tracer response measurements by injecting mono(galacturonic acid) as a tracer, those in the reactor were estimated to be a few seconds. PGA and bis- and tris(galacturonic acid)s were obtained from the supplier (Sigma-Aldrich Japan, Tokyo, Japan) and mono(galacturonic acid) and furfural from Wako Chemical Co. (Tokyo, Japan). These chemicals were used as received. The content of PGA in the solid sample was determined to be 94% by measuring the
yields of monomer, dimer, and trimer obtained by enzymatic hydrolysis of the sample. The products were identified by comparing the retention times between the standard sample and the product peaks in HPLC chromatograms and confirmed with an electrospray time-of-flight (TOF) mass spectrometer (model Micromass-LCT, Micromass; Jasco, Tokyo, Japan). The monomer and oligomers were quantitatively measured by a HPLC equipped with UV and refractive index detectors by using two columns (GS220HQ and SH1821 columns; Shodex, Tokyo, Japan) connected in series. The peak signals were collected through the chromatographic data processing software by a personal computer. The content of total organic carbon (TOC) was measured by a total carbon (TC) analyzer (model 5000A; Shimadzu, Kyoto, Japan). To obtain total yields of water-soluble oligomers, enzymatic hydrolysis of the eluted products was made with pectinase (P4300; Sigma-Aldrich Japan): One unit of pectinase (approximately 1.4 mg) was dissolved with 2 mL of 0.05 N ammonium acetate, adjusted to pH ) 4, in a vial. A total of 0.4 mL of the sample solution was put in the vial, stirred gently for 6 h at room temperature, and then was analyzed by HPLC. The product solution was allowed to settle for 2 h at room temperature, and the solid particles became precipitated. The particles were separated by filtration with a 0.22 µm membrane filter and then dried at 60 °C. Infrared spectra of the solid products were measured with a KBr disk by a Fourier transform infrared spectrometer (FTIR 600; Jasco, Tokyo, Japan) and scanned from 4000 to 400 cm-1 100 times at a resolution of 4 cm-1. Results and Discussion Figure 4 plots the effect of temperature on TOC recovered, which was the total amount of TOC dissolved in the solution. The recovered TOC values slightly increased with temperature up to 493 K, and these decreased with increasing temperature above 493 K. At higher temperatures, the formation of char and further decomposition of the products seemed to take place slightly. Note that the formation of gaseous products was less likely. In the temperature range studied except at 453 K, the components higher than about 90% of the initial solid sample was decomposed and/or dissolved with water within 2 min. Major products in the eluted aqueous solution were mono(galacturonic acid) and its oligomers. The HPLC chromatogram showed the peaks of galacturonic acid monomer and its oligomers from DP ) 2 to 10. The product solutions were also analyzed with the TOF mass
2312 Ind. Eng. Chem. Res., Vol. 43, No. 10, 2004 Table 1. Yields of Products and TOC Eluteda temperature, K 453
Figure 5. Cumulative product yields and TOC value vs time at 493 K: (3) TOC; (O) total yields of water-soluble oligomers, including monomer; (0) total yields of oligomers with DP > 10; (+) total yields of monomer and oligomers with DP ) 2-10; (*) monomer; (4) dimer; (×) trimer; (]) furfural.
spectrometer, and the oligomers up to DP ) 8 were detected. Furfural was considered to be the secondary product, which was produced from further decomposition of the monomer and oligomers, and was also detected by HPLC. Note that yields of the oligomers with DP ) 4-10 were estimated from the calibration obtained with tris(galacturonic acid) because the standard samples with DP ) 4-10 were not available. Figure 5 plots cumulative TOC values and cumulative yields of major products vs heating time at 493 K. The major dissolution step was terminated up to 2 min, while the furfural yield gradually increased with time up to 4-6 min. The value of TOC reached nearly 100% of the organic carbon content in the initial sample. However, each yield of monomer, dimer, or trimer was less than 10% on the carbon weight basis, and the yield of monomer plus oligomers with DP up to 10 was 30.1%. The remainder could be oligomers with DP higher than 10. The furfural yield was as much as 0.47%. Table 1 lists the product distribution and TOC content in the solution at various temperatures. The yields indicated were the cumulative values up to 15 min at each temperature. The monomer yields were higher than the oligomer yields at every temperature. The yield substantially decreased with the DP value, while the dimer yield was sometimes lower than the trimer yield. The yields of monomer and dimer increased with increasing temperature, while the yields for oligomers with DP higher than 4 decreased. The total yields of monomer and oligomers with DP ) 2-10 reached the maximum value at 533 K. The furfural yield was always lower than 1 wt %. The total yield of monomer and oligomers, including DP > 10, at 533 K was the lowest. This may be caused by further decomposition of products. Figure 6 plots the relationships between cumulative oligomer yields vs cumulative monomer yields for (a) dimer and (b) trimer at various temperatures. As depicted, the yields for dimer and trimer were nearly proportional to the monomer yield at lower monomer yields, irrespective of temperature. The slopes were 1.1 for dimer and 1.2 for trimer. At temperatures higher than 493 K and higher monomer yields, the yields of both dimer and trimer were more deviated from the linear relationships because of further decomposition. The furfural yield is plotted vs monomer yield in Figure 7. In plots up to 6-10% of monomer yield, the
453
493
monomer and oligomer yields, 100 × g of C/g of C of the initial sample DP ) 1 4.32 4.35 6.94 DP ) 2 4.14 4.49 5.71 DP ) 3 4.75 3.96 6.39 DP ) 4 3.65 2.92 4.63 DP ) 5 2.56 2.11 3.18 DP ) 6 1.41 1.19 1.76 DP ) 7 0.70 0.61 0.87 DP ) 8 0.33 0.29 0.40 DP ) 9 0.12 0.12 0.14 DP ) 10 0.04 0.04 0.05 total yield of oligomers 22.0 20.1 30.1 (DP ) 1-10) furfural yield, 100 × g of C/g of C 0.17 0.35 0.47 of the initial sample TOC, 100 × g of C/g of C 96.9 101.7 98.5 of the initial sample total yield of monomer 73.3 87.8c 78.5 and oligomersb
533
11.36 6.88 6.30 4.51 2.39 1.17 0.52 0.22 0.07 0.01 33.4 0.98 93.1 59.1
a All yields were cumulative values up to 15 min. b Total yields of the monomer, the dimer, and the trimer were obtained from enzymatic hydrolysis of water-soluble oligomers produced. c Enzymatic hydrolysis of water-soluble and solid products.
Figure 6. Yields of (a) dimer and (b) trimer vs monomer yield at various temperatures: (0) 453 K; (5) 473 K; (O) 493 K; (+) 503 K; (7) 513 K; (×) 523 K; (]) 533 K.
yield was represented by a straight line with a slope of 0.025, irrespective of temperature. At monomer yields higher than a certain value at each temperature, the corresponding furfural yield rapidly increased, although the monomer yield no longer increased. The threshold monomer yield, at which the furfural yield started rapid increasing, increased with temperature. Figure 8 shows yields of the monomer, the dimer, and the trimer obtained from enzymatic hydrolysis of com-
Ind. Eng. Chem. Res., Vol. 43, No. 10, 2004 2313
Figure 7. Cumulative furfural yield vs cumulative monomer yield at various temperatures. The legend is the same as that in Figure 6.
Figure 8. Cumulative yields of the monomer, the dimer, and the trimer obtained from enzymatic hydrolysis of water-soluble monomer and oligomers produced at 493 K.
ponents dissolved in the product solution eluted at 493 K, which involved 98.5% of organic carbon in the initial sample (see Table 1), and the yield of components of DP ) 1-10 was 30.1% on the carbon weight basis. The eluted component was fully hydrolyzed by the enzyme and converted to lower molecular components (DP ) 1-3): the yield of monomer was 33.4% on the carbon weight basis of the initial sample, and those of dimer and trimer were 39.2 and 6.2%, respectively. For infrared spectra of the filtrated components, the characteristic bands at 1100 and 1018 cm-1 as a fingerprint for pectic polysaccharide12 were observed for the products at 473 K, but those were smaller at higher temperatures. This indicates that products at higher temperatures were deformed slightly. At higher temperatures, more yields of monomer and oligomers with DP ) 2-10 resulted, while the degree of the deformation for higher DP components, which were precipitated after 2 h of settlement, increased. Figure 9 indicates the relationships between the cumulative TOC value and (a) total yields of monomer plus oligomers with DP ) 2-10 and (b) the total yields of the monomer, the dimer, and the trimer after enzymatic hydrolysis of water-soluble oligomers produced. Without further enzymatic hydrolysis treatment, the total yield of monomer and oligomers (DP ) 2-10) was roughly proportional to the cumulative TOC value at each temperature, but the slope became steeper at higher temperatures. After enzymatic hydrolysis of eluted products, the total yield was proportional to the
Figure 9. Cumulative yields of monomer and oligomers vs TOC recovered at various temperatures: (a) monomer and oligomers with DP ) 2-10; (b) those of water-soluble monomer and oligomers produced, which were enzymatically hydrolyzed into monomer, dimer, and trimer. The legend is the same as that in Figure 6.
TOC value and irrespective of temperature in the range of the major dissolution, namely, up to the dissolution of 75-80% of organic carbon content in the initial sample. Above 75-80% of TOC values, the plots showed the plateau. This implies that further decomposition of the major products took place at higher TOC values while that was insignificant in the major dissolution range. In comparison with the results shown in both figures, the eluted components involved a large amount of oligomers having DP > 10. Conclusions PGA was converted to the monomer and its oligomers by water in a semibatch flow reactor without any additives in the temperature range from 453 to 533 K at 10 MPa. The highest yield of monomer was obtained to be 11.4% on carbon weight basis within 2 min at 533 K of the highest temperature studied, and correspondingly those of the dimer, the trimer, and components having DP ) 4-10 were 6.9, 6.3, and 8.9%, respectively. However, the infrared spectra of precipitated components indicated that some structural deformation could take place somewhat at the temperature. At 493 K, the yields of components with DP ) 1-10 were lower than those at 533 K, and more than 90% of the TOC in the initial sample was dissolved and degraded into lower DP components. Acknowledgment The authors are grateful to the Promotion and Mutual Aid Corp. for Private Schools of Japan for a project
2314 Ind. Eng. Chem. Res., Vol. 43, No. 10, 2004
research grant (Development of Molecular Functional Materials in 2000-2002). Literature Cited (1) Yen, G. C.; Lin, H. T. Effects of High Pressure and Heat Treatment on Pectic Substances and Related Characteristics in Guava Juice. J. Food Sci. 1998, 63, 684. (2) Plat, D.; Ben-Shalom, N.; Levi, A.; Reid, D.; Goldschmidt, E. E. Degradation of Pectic Substances in Carrots by Heat Treatment. J. Agric. Food Chem. 1988, 36, 362. (3) Coimbra, M. A.; Barros, A.; Barros, M.; Rutledge, D. N.; Delgadillo, I. Multivariate Analysis of Uronic Acid and Neutral Sugars in Whole Pectic Samples by FT-IR Spectroscopy. Carbohydr. Polym. 1998, 37, 241. (4) Renard, C. M. G. C.; Cre´peau, M. J.; Thibault, J. F. Structure of the Repeating Units in the Rhamnogalacturonic Backbone of Apple, Beet and Citrus Pectins. Carbohydr. Res. 1995, 275, 155. (5) Kravtchenko, T. P.; Penci, M.; Voragen, A. G. J.; Pilnik, W. Enzymic and Chemical Degradation of Some Industrial Pectins. Carbohydr. Polym. 1993, 20, 195. (6) Leita˜o, M. C. A.; Silva, M. L. A.; Januario, M. I. N.; Azinheira, H. G. Galacturonic Acid in Pectic Substances of Sunflower Head Residues: Quantitative Determination by HPLC. Carbohydr. Polym. 1995, 26, 165. (7) Nothnagel, E. A.; McNeil, M.; Albersheim, P.; Dell, A. Hostpathogen Interactions XXII. A Galacturonic Acid Oligosaccharide from Plant Cell Walls Elicits Phytoalexins. Plant Physiol. 1983, 71, 916. (8) Grohmann, K.; Baldwin, E. A.; Buslig, B. S.; Ingram, L. O. Fermentation of Galacturonic Acid and Other Sugars in Orange
Peel Hydrolyzates by the Ethanologenic Strain of Escherichia coli. Biotechnol. Lett. 1994, 16, 281. (9) Mok, W. S. L.; Antal, M. J. Uncatalyzed Solvolysis of Whole Biomass Hemicellulose by Hot Compressed Liquid Water. Ind. Eng. Chem. Res. 1992, 31, 1157. (10) Bonn, G.; Concin, R.; Bobleter, O. HydrothermolysissA New Process for the Utilization of Biomass. Wood Sci. Technol. 1983, 17, 195. (11) Marshall, W. L.; Franck, E. U. Ion Product of Water Substance, 0-1000 °C, 1-10,000 Bars New International Formulation and Its Background. J. Phys. Chem. Ref. Data 1981, 10, 295. (12) Mochidzuki, K.; Sakoda, A.; Suzuki, M.; Izumi, J.; Tomonaga, N. Structural Behavior of Rice Husk Silica in Pressurized Hot-Water Treatment Processes. Ind. Eng. Chem. Res. 2001, 40, 5705. (13) Sasaki, M.; Kabyemela, B.; Malaluan, R.; Hirose, S.; Takeda, N.; Adschiri, T.; Arai, K. Cellulose Hydrolysis in Subcritical and Supercritical Water. J. Supercrit. Fluids 1998, 13, 261. (14) Sakaki, T.; Shibata, M.; Miki, T.; Hirose, H.; Hayashi, N. Reaction Model of Cellulose decomposition in Near-critical Water and Fermentation of Products. Bioresour. Technol. 1996, 58, 197. (15) van Walsum, G. P.; Allen, S. G.; Spencer, M. J.; Laser, M. S.; Antal, M. J.; Lynd, L. R. Conversion of Lignocellulosics Pretreated with Liquid Hot Water to Ethanol. Appl. Biochem. Biotechnol. 1996, 57/58, 157.
Received for review April 11, 2002 Revised manuscript received October 8, 2003 Accepted November 6, 2003 IE0202672
