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Autohydrolysis of Almond Shells for the Production of Xylo-oligosaccharides: Product Characteristics and Reaction Kinetics Debora Nabarlatz, Xavier Farriol, and Daniel Montane´ * Department of Chemical Engineering, ETSEQ, Rovira i Virgili University, Av. Paı¨sos Catalans 26, E-43007 Tarragona (Catalunya), Spain
Almond shells are agricultural residues with a high content of xylan that are produced abundantly in some regions with a Mediterranean climate. We have studied the production of xylo-oligosaccharides from almond shells by autohydrolysis at 150-190 °C. The yield, composition, and molar mass distribution of the xylo-oligosaccharides were dependent on temperature and time: the maximum yield of xylo-oligosaccharides increased from 42% at 150 °C and 300 min to 63% at 190 °C and 19 min, while their anhydroarabinose-to-anhydroxylose and acetyl-toanhydroxylose mass ratios were 0.039 and 0.076 at 150 °C and 0.129 and 0.125 at 190 °C, respectively. Experimental data was used to fit the parameters of a kinetic model for xylan autohydrolysis, which describes the yields of the different reaction products and accounts for the changes in the chemical composition of xylan and xylo-oligosaccharides. The recovery of the xylo-oligosaccharides by spray drying was also evaluated. Spray drying of the autohydrolysis liquor obtained at 179 °C and 23 min gave a yield of nonvolatile products of 29.4 g/(100 g of dry almond shells). The composition of this product was 58.3% xylo-oligosaccharides, 2.4% xylose, 1.5% arabinose, 0.78% glucose, 0.27% HMF, 16% Klason-type lignin, 4.8% ash, and 14.9% nonidentified products, which probably included organic extractives and protein present in almond shells, and products formed by the degradation of carbohydrates. Introduction Xylo-oligosaccharides derived from xylan-rich hemicelluloses are carbohydrates with a high potential for novel applications in the chemical, food, and pharmaceutical industries. Ethers and esters prepared from xylan and xylo-oligosaccharides have been synthesized and used as thermoplastic compounds for biodegradable plastics, water soluble films, coatings, capsules, and tablets,1 and also for the preparation of chitosan-xylan hydrogels.2 In the food industry, xylo-oligosaccharides can be used as low-calorie sweeteners and as soluble dietary fiber since they are not metabolized by the human digestive system. However, they act as prebiotics, providing a source of carbon for the development of intestinal microflora and probiotic microorganisms,3-6 and are already used in fortified foods aimed at the development of intestinal microflora.7,8 Besides, xylooligosaccharides have acceptable organoleptic properties and do not exhibit toxicity or negative effects on human health. Another area that may provide xylan and xylooligosaccharides with applications is the synthesis of active compounds against viral and carcinogenic processes, which have already been synthesized from multiple hemicellulose-derived carbohydrates. The activity of these substances depends largely on the structure of the polysaccharide. Sulfates prepared from xylan and other polysaccharides modulate the function of proteins and some processes at the cell-wall level and possess antiviral9-11 and antitumoral12-14 activity. Recently, xylo-oligosaccharides extracted by autohydrolysis of bamboo have been found to possess a cytotoxic effect on human leukemia cells.15 * Corresponding author. Tel.: (+34) 977 559 652. Fax: (+34) 977 558 544. E-mail:
[email protected].
Most commercial xylo-oligosaccharides are produced by the enzymatic hydrolysis of xylan that has been isolated from biomass by extraction with potassium hydroxide. Because of the extraction procedure, those xylo-oligosaccharides lose acetyl groups and uronic acids by saponification and have very limited solubility in neutral aqueous solutions. Autohydrolysis of lignocellulosic biomass is an efficient process to produce xylooligosaccharides with a reasonable yield and a wide variety of compositions. Since autohydrolysis takes place in slightly acidic media, part of the side groups in the backbone xylose chains, like acetyl, uronic acids, and phenolic acid substituents, remain in the xylo-oligosaccharides.16,17 These are highly soluble in water and show a behavior during enzymatic hydrolysis that is completely different from that of xylo-oligosaccharides isolated by alkaline extraction.17 The differential characteristics of the xylo-oligosaccharides obtained by autohydrolysis versus those obtained from alkali extraction has prompted a renewed interest in developing process strategies to achieve a high yield of xylo-oligosaccharides with consistent reproducibility in purity, composition, and molar mass distribution.18,19 In this paper, we have explored the production of xylo-oligosaccharides from almond shells, a xylan-rich agriculture residue. Experiments were developed in 25 mL tubular batch reactors operated at 150, 169, and 190 °C and a solid-to-liquid mass ratio of 1/8. These experiments provided information on the yield and composition of xylan in the hydrolyzed solid, the xylo-oligosaccharides that are formed as primary products, and the molar mass distribution of the xylooligosaccharides. Experimental data was used to fit the parameters of a kinetic model for xylan hydrolysis. The robustness of the model was tested by comparing
10.1021/ie050664n CCC: $30.25 © 2005 American Chemical Society Published on Web 08/27/2005
Ind. Eng. Chem. Res., Vol. 44, No. 20, 2005 7747 Table 1. Average Chemical Composition and Confidence Interval (r ) 0.025) of the Batch of Almond Shells Used for This Study composition (% dry basis)
average
confidence interval
ash EtOH/toluene extractives Klason lignin glucana xylana arabinana acetyl other (by difference) arabinan/xylan (mol/mol) acetyl/xylan (mol/mol)
2.83 5.0 27.4 26.8 26.1 2.4 4.0 5.5 1:9.3 1:15.2
0.07 0.4 1.4 0.6 0.8 0.3 0.9
a
Anhydrous monosaccharide.
calculations for the autohydrolysis of almond shells at 179 °C with results from additional experiments in a 10 L stirred batch reactor. Finally, the recovery of the xylo-oligosaccharides by spray drying and the purity and molar mass distribution of the recovered product were also studied.
Figure 1. Temperature profiles for replicate experiments at a nominal temperature of 179 °C: tubular batch reactor (top, triplicate) and 10 L stirred batch reactor (bottom, quadruplicate).
Experimental Section Materials. Autohydrolysis of almond shells was studied using homogeneous samples taken from a batch of 100 kg of almond shells ground and sieved to 300 µm. The batch was purchased from MIMSA S. A. (Lleida, Spain). It had a moisture content of 11% on receipt and was used without any further treatment. Table 1 shows the chemical composition of the ground almond shells, which was determined according to the analytical procedures described below. Reactor Systems. Two reactors systems of different scales were used in this study, 25 mL tubular batch reactors and a 10 L stirred batch reactor. The tubular batch reactors were constructed using 25 mm o.d. stainless steel bulkhead union fittings (Let-Lok 774L, Ham-Let, U.K.). One end of the fitting was capped with a 25 mm stainless steel plug (Let-Lok 7121L, Ham-Let, U.K.), and the other end mounted a combination of stainless steel union-reductions (Let-Lok 767LT, HamLet, U.K.) to accommodate a 1.5 mm K-type thermocouple (RS-Amidata, Barcelona, Spain) mounted inside the reactor to record the temperature-time history of each experiment. The reactors were mounted horizontally on a support and heated by immersion in a stirring thermal-oil bath (Digiterm 200, Selecta, Spain), which was previously adjusted to the desired reaction temperature. At the end of the reaction period, the reactors were removed from the oil bath and rapidly submerged in water at room temperature to quench the reaction. Figure 1 shows the typical temperature-time profiles in this system for a triplicate experiment at 179 °C. The complete operation procedure and has been reported previously.20 A total of 46 experiments were performed at three temperatures (150, 169, and 190 °C) and reaction times up to 330 min. All experiments were performed at a nominal concentration of dry almond shells of 11%. When the reactor was cold, it was opened and the reaction products were filtered to recover the hydrolyzed solid. This was washed with warm water (three consecutive washes with 15 mL) and dried at 105 °C. The washings were collected with the liquid from the reactor, measured, and stored for further analysis. The 10 L stirred batch reactor was constructed of ANSI 304-L and 316-L stainless steel by EMMSA (Tarragona, Spain), and it was equipped with a variable
speed Magnedrive II stirrer (Autoclave Engineers, U.S.), four internal baffles to improve mixing, an internal coil for heating and cooling, and a K-type thermocouple for temperature acquisition along the experiment. Heating was achieved by flowing saturated steam through the inner coil at a pressure adequate to maintain the desired temperature inside the reactor. When the target reaction time was reached, the flow of steam was stopped and tap water was circulated through the inner coil to quench the reacting mixture. Figure 1 shows the temperature profiles for four experiments at 179 °C. The heating rate is almost identical to that obtained in the tubular batch reactors, but the cooling rate was lower. Once cooled, the reactor was depressurized through a valve on the cover and it was emptied into a 50 L polypropylene container through a 1 in. ball valve located at the bottom of the vessel. The reactor was thoroughly washed with deionized water, and the washings were mixed with the product. The hydrolyzed solid was then separated by filtration, washed, and dried at room temperature. The yield of hydrolyzed solid was calculated from the weight of the whole solid and its moisture content, which was determined by drying three samples at 105 °C until a constant weight. The liquid product was measured, and a 100 mL sample was taken for analysis. The rest of the liquid product was either treated by spray drying to recover the xylo-oligosaccharides or frozen and stored for further processing. Four experiments were performed at 179 °C for 23 min in the 10 L reactor to compare the results with those calculated from the kinetic model, which was adjusted using data from the tubing-bomb reactor system. The experiments were performed at a dry almond shells concentration of 14.3%. Spray Drying. The xylo-oligosaccharides in the liquid product from the autohydrolysis experiments in the 10 L reactor were recovered by atomization using a Bu¨chi B-290 spray dryer (purchased from Masso Analı´tica, Barcelona, Spain) operated at a feed flow rate of 8 mL/min, an outlet temperature of 85 °C, and an air flow rate of 670 L/h. Preliminary tests with dextran solutions of known concentration and molar mass distributions close to those of the xylo-oligosaccharides showed that the dextrans were recovered quantitatively.
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Figure 2. Amount and composition of the nonreacted xylan remaining in the hydrolyzed almond shells at 150 °C (0, - - -), 169 °C (3, ‚‚‚), and 190 °C (O, s). Lines show the values calculated with the kinetic model using the best-fit parameters. The amount of xylan is expressed as a percentage of the xylan in native almond shells, while the composition is expressed as a mass fraction of the remaining xylan.
Figure 3. Yield and composition of the xylo-oligosaccharides at 150 °C (0, - - -), 169 °C (3, ‚‚‚), and 190 °C (O, s). Lines show the values calculated with the kinetic model using the best-fit parameters. The yield of xylo-oligosaccharides is expressed as a percentage of the xylan in native almond shells, while the composition is expressed as a mass fraction of the xylo-oligosaccharides.
Analytical Methods. The hydrolyzed almond shells were analyzed for carbohydrates, acetyl groups, and Klason lignin (ASTM D 1106-84). Analysis was performed by quantitative hydrolysis of the solid in 72% sulfuric acid at 30 °C for 60 min. The solution was then diluted to 4% sulfuric acid, and the reaction was continued at 120 °C for 45 min. A sample of this acidic solution was then filtered through a 0.22 µm nylon syringe filter (Tracer 13 mm, Teknokroma, Spain) and analyzed by HPLC, as described below. The raw almond shells were analyzed using the same procedure, but using samples that were extractive-free. Extractives were removed with an ethanol-toluene mixture according to a modification of the ASTM D 1107-84 standard method in which benzene is replaced by toluene. The percentage of ash in the original, nonextracted material was measured by the ASTM D 1102-84 standard method. The liquid product from the reactor (reaction liquid + washings) was also analyzed. A sample of 2 mL was filtered through a 0.22 µm syringe filter and analyzed by HPLC to measure the amounts of monosaccharides, acetic acid, furfural, and hydroxymethylfurfural (HMF). Another sample of 5 mL was taken and
mixed with 1 mL of H2SO4 (5N). The acidified solution was hydrolyzed at 120 °C for 45 min to convert all oligosaccharides into their constitutive monomers. The solution was then filtered through a 0.22 µm syringe filter, and monosaccharides and acetic acid were quantified by liquid chromatography. HPLC analyses were performed with an Agilent 1100 series chromatograph (purchased from Agilent, Barcelona, Spain) using a BioRad HPX 87H column (Bio-Rad Laboratories, U.S.) at 30 °C. The mobile phase was 0.005 M H2SO4 at a flow rate of 0.5 mL min-1. An Agilent 1100-DAD ultraviolet diode-array (UV) detector and an Agilent 1100-RID refractive index (RI) detector were connected in series. The UV detector was used to quantify furfural and HMF in the samples that contained low concentrations of these compounds. The RI detector was used for the samples with high concentrations of furfural and HMF and for quantifying acetic acid and carbohydrates. The HPLC system was calibrated with standards of known concentration prepared from xylose (>99.5%, SigmaAldrich), glucose (>99,6%, Fluka), arabinose (>99.0%, Fluka), furfural (>99.0%, Fluka), HMF (>95.0%, Fluka), and acetic acid (>99.7%, Panreac, Spain).
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A third sample of the autohydrolysis liquor was taken for analyzing the molar mass distribution of the soluble products by gel permeation chromatography (GPC). The sample was mixed with a solution of KNO3 and sodium azide, to adjust the concentrations of KNO3 and sodium azide to 0.05 mol/L and 83 mg/mL, respectively, the same values that were used in the solvent of the GPC analysis. This avoided the appearance of negative peaks in the chromatogram. The analyses were carried out in the HPLC (high-performance liquid chromatography) chromatograph mentioned above, using the GPC addon of the LC Chemstation software (purchased from Agilent, Barcelona, Spain). The analyses were performed in a TSKGel G3000PWXL column (Toso Haas, purchased from Teknokroma, Barcelona, Spain) at 25 °C using 0.5 mL/min of a 0.05 mol/L solution of KNO3 with 83 mg/L of sodium azide as solvent, and using the refractive index detector. The GPC system was calibrated with xylose, glucose, and low polydispersity standards of maltose oligomers and dextrans (Fluka). Results and Discussion Xylan Conversion and Yield of Xylo-oligosaccharides. Xylans are polyoses that are formed by a homopolymer backbone of xylose units. Several functional groups (uronic acids, arabinose, galactose, and acetyl and ferulic acids) can be attached to the xylan backbone in variable amounts, which results in the large diversity of xylan structures found in plants.21,22 In this work, we have considered xylan to be a polymer made up of three constitutive monomers: xylose, arabinose, and acetic acid. Xylan from almond shells should contain variable amounts of glucuronic and ferulic acids as well, but we could not measure them accurately and they were not included in our results. Consequently, the quantity of xylan in the hydrolyzed solid and the yield of xylo-oligosaccharides were calculated from the amounts of anhydrous xylose, arabinose, and acetic acid determined by quantitative hydrolysis and HPLC analysis. Figure 2 shows the xylan that remains in the hydrolyzed almond shells at the three temperatures we tested, expressed as a fraction of the initial xylan in the untreated almond shells. Xylan decreased to 35% of the initial composition in 335 min at 150 °C, while it took only 13 min to reach the same conversion at 190 °C. After 30 min at the latter temperature, only 15% of the initial xylan remained in the hydrolyzed solid. The composition of the xylan changed as the hydrolysis progressed. In general, the mass fraction of xylose in xylan increased while that of arabinose decreased. Acetyl groups reached a maximum at an intermediate hydrolysis time. The yield of the xylo-oligosaccharides measured in the aqueous phase at the three reaction temperatures is shown in Figure 3, expressed as a percentage of the xylan in the untreated almond shells. The maximum yield at 150 °C was ∼42% after 300 min of hydrolysis. Higher temperature favored an increase in the yield of xylo-oligosaccharides, which reached a maximum of 63% at 190 °C and 19 min of hydrolysis. The chemical composition of the xylo-oligosaccharides was also influenced by temperature and time. Those produced at short reaction time had a high content of arabinose, but it decreased to much lower values after a few minutes of hydrolysis. The content of xylose increased steadily with reaction time, and acetyl groups reached a maximum along hydrolysis.
Figure 4. Change in the molar mass distribution of the xylooligosaccharides during autohydrolysis at 150 °C (top), 169 °C (middle), and 190 °C (bottom). Areas of the distribution are proportional to the yield on xylo-oligosaccharides.
Figure 4 shows the molar mass distribution of the xylo-oligosaccharides at selected times of hydrolysis for experiments at 150, 169, and 190 °C. The normalized areas of the molar mass distribution plots were multiplied by the yield of xylo-oligosaccharides of each experiment for better interpretation. The GPC analysis only provided a qualitative description of the molar mass distribution of the xylo-oligosaccharides since it was performed on samples of the hydrolysis liquid and it accounts for all soluble products. This includes xylooligosaccharides, but it also inlcudes monosaccharides and their degradation products, lignin-derived products, and other constituents of the almond shells such as
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Figure 5. Yields of xylose, acetic acid, arabinose, and furfural, and the amount of degraded xylan at 150 °C (0, - - -), 169 °C (3, ‚‚‚), and 190 °C (O, s). Lines show the values calculated with the kinetic model using the best-fit parameters. The yield of products is expressed as a percentage of the xylan in native almond shells.
inorganic salts, organic extractives, and minor amounts of protein and pectin, which are solubilized during hydrolysis. The latter are present in almond shells at typical levels of 1.0% and 0.58%, respectively.23 At the beginning of the hydrolysis process, the soluble products comprised a wide range of molar masses. After 7.8 min at 150 °C, the xylo-oligosaccharides had a very wide mass distribution that covered from 0.1 to 1000 kDa and showed four characteristic peaks at 248, 3.9, 1.6, and 0.235 kDa. The high molar mass peak at 248 kDa decreased because of the hydrolysis of the solubilized xylo-oligosaccharides as the reaction proceeded, but there were some oligomers of molar mass >200 kDa present even at a very long reaction time (230 min at 150 °C). This means that a small fraction of high molar mass xylo-oligosaccharides was formed during most of the extent of the reaction. However, the molar mass distribution of the oligomers narrowed as the hydrolysis advanced. At 150 °C and 95 min, most of the xylo-oligosaccharides had a molar mass
