688
Ind. Eng. Chem. Res. 1997, 36, 688-696
Hydrolytic Pretreatment of Softwood and Almond Shells. Degree of Polymerization and Enzymatic Digestibility of the Cellulose Fraction Jose´ Miguel Martı´nez, Jordi Reguant, Miguel A Ä ngel Montero, Daniel Montane´ , Joan Salvado´ , and Xavier Farriol* Departament d’Enginyeria Quı´mica, Escola Te` cnica Superior d’Enginyeria Quı´mica, Universitat Rovira i Virgili, Autovia de Salou S/N, 43006 Tarragona, Catalunya, Spain
Autohydrolysis and dilute-acid hydrolysis were used as pretreatment methods to enhance the enzymatic digestibility of cellulose in two different lignocellulosic substrates, a softwood mixture and an agricultural residue of almond shells. The changes in the chemical composition of the softwood mixture during dilute-acid pretreatment were studied, and the results were grouped by using a severity parameter, KROH, which was derived from the kinetics of hemicellulose solubilization. The average degree of polymerization of the cellulose retained in the pretreated substrate, DPv, was investigated and its trend compared with that of almond shells. Cellulose DPv decreased for both the substrates from a value of 700 for the untreated lignocellulosic to around 200, which corresponds to the leveling-off degree of polymerization, as there is no significant variation in DPv when severity increases. Glucose yields after enzymatic saccharification of the pretreated substrates turned out to be higher for almond shells than for the softwood mixture, which had a very low susceptibility to enzymatic hydrolysis throughout the spectrum of experimental conditions investigated. At KROH ) 15 000, the glucose yield for the softwood mixture is 44% of the potential fraction present in the pretreated pulp, while for almond shells the maximum yield surpasses 97% for KROH ) 3. The different behavior between these lignocellulosic species is not caused by different degrees of cellulose depolymerization nor by the removal of different amounts of carbohydrates during pretreatments in equivalent conditions. Introduction Cellulose is the largest wood component, making up approximately half of both softwoods and hardwoods. It can be briefly characterized as a linear high-molecular-weight polymer built up exclusively of β-D-glucose units. Major uses of cellulose are the production of paper and related products and the manufacture of rayon and other derivatives such as cellulose esters and cellulose ethers from high-purity cellulose preparations. Cellulose can also be hydrolyzed to glucose. The production of glucose is the first and most important step in the conversion of cellulose to low-molecular-weight compounds and a wide range of different chemicals such as acetone, alcohols, organic acids, and yeast (Fengel and Wegener, 1984). The conversion of cellulose into cellulose derivatives is based on a number of physical and chemical pretreatment methods which partially depolymerize cellulose, enhancing its reactivity to chemicals and enzymes and enabling it to be purified. Some of these pretreatment methods are physical (milling, irradiation), while others are chemical (delignification, hemicellulose removal, cellulose dissolution, and reprecipitation), or combinations of the above (Grethlein, 1984). The aim of this paper is to describe how the cellulose fraction in two different substrates, almond shells and a softwood mixture, is modified during hydrolytic pretreatment. Hydrolytic pretreatment is the key stage in determining the extent of the chemical and structural modifications experienced by the lignocellulosic material in the fractionation process, which pursues the separation of the main polymeric components (i.e., cellulose, * Author to whom all correspondence should be addressed. e-mail:
[email protected]. S0888-5885(96)00048-6 CCC: $14.00
lignin, and hemicelluloses) at the maximum yield while preserving their macromolecular and chemical properties. The viscosity-average degree of polymerization of cellulose, DPv, and the yield of glucose obtained by enzymatic saccharification of the pretreated substrate have been studied in different pretreatment conditions. The pretreatment procedure is based on a controlled hydrolytic depolymerization in aqueous slurry, which is catalyzed either by the acidic species in wood (autohydrolysis) or by addition of mineral acids (prehydrolysis) (Springer, 1966; Springer and Zoch, 1968; Grethlein, 1978; Maloney et al., 1986; Overend and Chornet, 1987). Pretreatments were performed in an isothermal continuous tubular reactor and in a nonisothermal batch stirred reactor. The main operation variables involved in the pretreatment, temperature, residence time, and acid concentration were unified with a severity parameter, which was used to model the kinetics of hemicellulose solubilization. Materials and Methods Lignocellulosic Substrates. Two different lignocellulosic substrates were used in this study, a homogeneous batch of 200 kg of a ground softwood mixture at 50% of spruce (Abies alba) and pine (Pinus insignis) and another 200 kg of an agricultural residue composed of almond shells. Almond shells are a lignocellulosic agriculture byproduct that has no established industrial application. Almond production is located mainly in areas with Mediterranean climates such as Spain, Italy, or California. There is a great diversity of almond species which exhibit different productivity and yields of seed in the fruit. Such a difference in the varieties means that the shell may be from 35 to 75% of the total © 1997 American Chemical Society
Ind. Eng. Chem. Res., Vol. 36, No. 3, 1997 689 Table 1. Average Composition and 95% Confidence Interval for the Softwood Mixture and Almond Shellsa
a
fraction
softwood mixture
confidence limit
almond shells
confidence limit
ash hot water extractives ethanol/etoluene extractives Klason lignin glucan xylan galactan arabinan mannan
0.4 7.4 3.3 25.1 38.2 5.9 4.1 6.6 12.0
0.1 1.4 1.4 0.8 0.7 0.3 0.2 0.3 0.8
0.6 4.1 0.5 28.3 28.0 30.2 0.5 2.2 1.7
0.1 0.4 0.1 0.6 0.6 0.4 0.2 0.3 0.3
Results expressed as percent of dry solid basis.
fruit weight (Saura Calixto et al., 1988). The physical properties of almond shells are different from those of hardwoods and softwoods because of their structural dissimilarities. Almond shells are harder and more rigid than wood, and also denser and less porous. Present world production of almond shells is about 550 000 tons yr-1 and is increasing. The high xylan content of the almond shell suggests that it is particularly suitable for producing xylose derivatives. So the two substrates, the softwood mixture and the almond shells, have in common their residual origin in commercial terms, their abundance in Catalonia, in the northeast of Spain, and their immediate disposability from commercial mills. A perfect mixed batch of a softwood mixture was ground and sieved to 100 mesh. The resulting sawdust had a moisture content of 7% of the total weight. The second substrate was a homogeneous batch of ground almond shells (Prunus amygdalus), which was sieved to 60 mesh and had a moisture content of 9% of the total weight. Table 1 shows the average composition of both substrates expressed in weight percent of the dry lignocellulosic (% DSB). Six and seven different samples were analyzed for almond shells and the softwood mixture, respectively. Analytical Procedures. Chemical analysis of the substrates and the pretreated samples was conducted using the following standard methods: ASTM E-87182 for moisture content; ASTM D-3516-76 for ash content; ASTM D-1111-84 for hot-water extractives; and modified ASTM D-1107-84 for ethanol/toluene extractives. Klason lignin was measured in the extractivefree samples using ASTM D-1106-84. Carbohydrates were analyzed by HPLC after the substrates had been quantitatively saccharified (Saeman et al., 1945). Conditions for the HPLC analysis have been detailed elsewhere (Montane´ et al., 1993). Holocellulose was prepared from an extractive-free sample by chlorite delignification (Browning, 1967a), and R-cellulose was isolated from holocellulose by extraction with concentrated sodium hydroxide according to the TAPPI T-203 05-74 method. The viscosity-average molecular weight and the degree of polymerization, DPv, of the cellulose remaining in the pretreated pulp were investigated in the R-cellulose fraction. The degree of polymerization was calculated from the intrinsic viscosity according to the relationship in eq 1 (Browning, 1967b). The intrinsic viscosity, [η]CED, was measured in a 0.5 M solution of cupriethylenediamine hydroxide (CED) using the ISO 5351/1-1981 standard.
DPv ) 1.90[η]CED
(1)
Pretreatment Conditions. Both pretreatments, dilute-acid hydrolysis and autohydrolysis, were con-
ducted in two continuous tubular reactors capable of processing aqueous suspensions of ground lignocellulosics. Two different configurations were used, a 2.6 m long reactor with an inner diameter of 28 mm, constructed in Hastelloy C, and a second reactor constructed in ANSI 316 stainless steel with a length of 45 m (three 15 m coils in series) and an inner diameter of 10 mm capable of reaching residence times higher than 5 min instead of the 1.2 min achieved with the first tubular reactor. The flow diagram of the process unit is shown in Figure 1. The slurry is prepared in a 55 L tank, T-1, which is equipped with four baffles and a stirring device, M-1, at 600 rpm (Dosapro Milton Roy DHR-B), and it is homogenized by rapid recirculation with the aid of an adjustable flow pump P-1 (Seepex 05-24BN). The pump P-2 (Dosapro Milton Roy MB) is used to preheat the reactor to the desired temperature using deionized water and a rapid steam injection from a fuel-oil fired boiler, SG (Sadeca SDE-75). The admission of either water or slurry is selected by means of valves V-1 and V-2 (Whitney SS-63TSW8P-133-SR). The slurry flow delivered to the reaction zone is also controlled by pump P-2. The system pressure is measured and recorded by an electronic transducer (Foxboro 841 GM-DI; range 0-4.9 MPass). Acids can be added to the slurry by a metering pump P-3 (Dosapro Milton Roy A 29-FR-125). After the acid injection point there is a static mixer, M-2, where the saturated steam is directly added to heat the slurry. The steam flow is regulated by valve V-3 (Masoneilan Varipak 28-2814011) driven by the reactor temperature via an actuator. Four thermocouples are installed along the tubular reactor. If an acid catalyst has been added to the slurry, it is neutralized by an alkali solution injected with pump P-4 (Dosapro Milton Roy A 29-FR-125). The treated slurry is flashed through valve V-4 to cool it and thus stop the hydrolytic reaction. After the flash there is a three-way valve which enables the product to be collected either in tank T-2, 80 L, used during the startup and shutdown procedures, or in tank T-3, 55 L, used to collect the material after it has been treated. The steam generated by the flash expansion through valve V-4 is condensed in a shell and tube heat exchanger, C-1. The average temperature of the reactor was assumed for calculations, while the residence time was calculated from the mass flow of hot slurry assuming that it had the density of water at the operating temperature. The average steam consumption was determined from a global mass balance (substract and water) during the period of product collection. In order to carry out the mass balance for the substract, the quantity of product fed was determined from the slurry flow register of pump P-2 and the solid concentration was determined by evaporating to dryness a minimum of three slurry samples taken at the beginning and at the end of the experiment. The amount of derived products collected
690 Ind. Eng. Chem. Res., Vol. 36, No. 3, 1997
Figure 1. Schematic diagram of the process unit used in this study.
in tanks T-2 and T-3 was calculated by measuring the quantity of insoluble, dry lignocellulosic fiber and soluble products by evaporation to dryness at 60 °C of various aliquots of the liquid phase. A total of 21 experiments (1-S to 22-S) were conducted for the softwood mixture in the second tubular reactor system using an average concentration of solids of around 7% (w/w). The experimental plan covered temperatures from 176 to 231 °C, residence times from 1.0 to 5.5 min, and acid loads from 0.0 to 4.5 g of sulfuric acid/kg of slurry. Eleven experiments (6-A to 16-A) were conducted for almond shells in the first tubular reactor system at temperatures ranging from 180 to 215 °C, residence times between 0.9 and 1.1 min, and acid loads from 0.0 to 2.2 g of sulfuric acid/kg of slurry. The concentration of lignocellulosics was maintained at 14% (w/w). Ten additional experiments (18-A to 28-A) were carried out for almond shells under autohydrolysis conditions in a 300 mL batch stirred reactor provided with three baffles (Autoclave Engineers E86-10271-1) at a speed of 500 rpm with electrical heating. The aim of these new runs was to study the variation of the constitutive polymers in the insoluble fraction at higher severities. In this series of experiments, 12 g of dry ground material was used and the concentration of solids was adjusted to 15% (w/w). Temperatures of 180 and 200 °C and reaction times from 1.0 to 44.0 min were used with an average heating rate from 5.0 to 10.3 °C/min and a cooling rate from 17.0 to 39.0 °C/min. At the end of the reaction the entire product was collected and filtered under vacuum. The diffusional limitations influence the hydrolysis mechanism. The solubilization of a high-molecularweight species will be strongly affected by the dynamic conditions in the aqueous phase. In experimental situations such as in the autoclave at 500 rpm in our case (Montane´ et al., 1993) or in the tubular reactor, where the lignocellulosic solids with a diameter of less than 0.5 mm are in contact with sufficient liquid water, the transport of solubilized polymers from the solid interface to the bulk liquid phase is increased by turbulent mass transfer and the solubilization process approaches a regime in which kinetic control may be assumed over diffusion control. So, no differences can
be observed in the evolution of constitutive polymers in the insoluble fraction (hemicellulose, lignin, and cellulose) when comparing different reactor configurations (autoclave and tubular reactor of 2.6 or 45 m long) if the suspension is well mixed. The flow regime is fully turbulent for the two tubular reactors considered, as Reynolds number is between 10 000 and 14 000 for the first configuration and between 16 000 and 23 000 for the second one if water properties are used. So, both the tubular systems are assumed to be plug flow reactors. Enzymatic Saccharification. The effect of the pretreatment on the susceptibility of the cellulose to enzymatic hydrolysis was studied in both substrates. Enzymatic hydrolysis was carried out using a cellulase complex from Aspergillus niger (Sigma C-2415). The pretreated lignocellulosics were suspended at 2% (w/w) solids in 2 mL of enzymatic solution (50 mM sodium citrate buffer at pH ) 4.8 with 0.005% sodium azide as a preservative). The samples were incubated in capped test tubes and Erlenmeyer flasks at 50 °C, with orbital shaking (200 rpm) for 48 h. The samples were then removed and centrifuged (3000 rpm for 5 min). Supernatants were analyzed for glucose with the glucose oxidase reaction. The load of lyophilized enzyme was 10 mg‚mL-1 in all the experiments. The activities of the system were 4.8 IU‚mL-1 filter paper (FP) activity, 28.3 IU‚mL-1 carboxymethylcellulose (CMC) activity, 19.6 IU‚mL-1 cellobiase activity, and 14.8 IU‚mL-1 β-glucosidase activity. Activities corresponding to FP, CMC, and cellobiose were determined as recommended by the IUPAC Commission for Biotechnology (Ghose, 1987). β-Glucosidase activity was evaluated as described by Kubicek (1983). Results and Discussion The operating conditions for pretreating softwood sawdust in the tubular reactor under autohydrolysis conditions are shown in Table 2, while Table 3 shows the results for the dilute-acid pretreatments. These tables also list the amount of solubilized lignocellulosic, the composition of the insoluble fraction (ethanol/ toluene-soluble lignin, Klason lignin, and anhydrous sugars), and the viscosity-average degree of polymeri-
Ind. Eng. Chem. Res., Vol. 36, No. 3, 1997 691 Table 2. Pretreatment of the Softwood Mixture by Autohydrolysis in the Continuous Tubular Reactor (Second Configuration)a experiment number experimental conditions temperature (°C) time (min) log(KROH) solubilization pulp composition anhydrous glucose other anhydrous sugars ethanol/toluene soluble lignin Klason lignin R-cellulose DPv
1-S
2-S
3-S
4-S
5-S
6-S
7-S
8-S
9-S
10-S
11-S
12-S
13-S
14-S
176 2.5 -0.04 17.5
187 3.1 0.08 23.8
191 3.1 0.11 26.0
198 2.9 0.15 28.6
215 3.0 0.27 29.3
217 2.8 0.28 31.4
227 2.0 0.30 30.5
225 2.9 0.34 31.4
223 4.3 0.39 32.1
231 3.0 0.39 33.1
221 5.4 0.41 32.3
227 4.8 0.43 34.8
229 4.8 0.45 35.5
231 5.5 0.48 39.2
35.6 17.7 3.3 24.0 630
34.8 13.3 2.5 23.8 510
34.6 12.9 2.1 23.7 610
36.0 10.9 2.5 22.9 530
36.6 10.6 3.0 21.4 450
37.7 9.0 5.0 21.1
34.7 9.5 2.4 21.7 480
37.2 8.7 5.7 20.7 330
37.4 8.3 3.7 20.5
33.5 8.4 3.5 20.0 410
38.0 7.9 3.2 21.8
38.8 5.0 4.0 19.9
34.5 5.6 4.7 19.2 400
33.1 4.4 4.2 18.2 330
a Operating conditions, yield, and chemical composition of the pretreated pulp. Results are expressed as percent of the initial dry solid (% DSB). Other anhydrous sugars include xylose, galactose, arabinose, and mannose.
Table 3. Pretreatment of the Softwood Mixture by Dilute-Acid Hydrolysis in the Continuous Tubular Reactor (Second Configuration)a experiment number experimental conditions temperature (°C) time (min) acid load (g of acid/kg of slurry) log(KROH) solubilization pulp composition anhydrous glucose other anhydrous sugars ethanol/toluene soluble lignin Klason lignin R-cellulose DPv
15-S
16-S
17-S
18-S
19-S
20-S
21-S
22-S
191 3.0 0.3 0.32 27.7
220 2.1 0.4 0.59 34.8
223 1.2 0.6 0.69 35.6
205 1.0 1.6 1.42 34.3
231 1.6 1.8 1.87 45.4
226 1.2 2.0 1.96 48.4
230 1.6 2.4 2.39 48.9
231 1.7 4.4 4.17 60.8
36.6 11.8 2.2 23.2 380
34.9 1.2 4.9 20.2 300
36.9 0.8 5.0 19.1 320
35.5 0.5 4.1 21.1 310
29.6 1.3 7.8 16.7 190
27.6 0.0 7.9 16.4 190
25.8 1.2 7.8 15.9 200
16.2 0.8 8.4 14.1 180
a Operating conditions, yield, and chemical composition of the pretreated pulp. Results are expressed as percent of the initial dry solid (% DSB). Other anhydrous sugars include xylose, galactose, arabinose, and mannose.
zation, DPv of the R-cellulose fraction. Experiments 1-S to 14-S, performed under autohydrolysis conditions, show the influence of the reaction temperature and the residence time on the yield and chemical composition of the pulp. The amount of solubilized material increases from 17.5% DSB (dry solid basis) at 176 °C and 2.5 min to 39.2% DSB at 231 °C and 5.5 min. Simultaneously, the amount of non-glucose sugars in the pulp decreases from 17.7% to 4.4% DSB in the same operating conditions. The content of anhydrous glucose remains approximately constant over the temperature range covered. The fraction of lignin which is soluble in ethanol/toluene increases slightly from 2% DSB at the lowest severity studied to 4.5% at the highest. On the other hand, the fraction of lignin which is insoluble in this mixture, measured as Klason lignin, decreases from 24% to 18.2% DSB in the aforementioned operating conditions, which indicates that a small fraction of the lignin originally present in the untreated softwood is solubilized during the autohydrolysis pretreatment. Experiments 15-S to 22-S show the effect of adding sulfuric acid at different combinations of reaction temperature and residence time. The addition of acid enhances solubilization, which increases from 27.7% to 60.8% DSB at the highest acid load used, 4.4 g of acid/ kg of slurry, or 10.4 g of acid/100 g of dry lignocellulosic. Hemicellulose is the main contributor to this increase in solubilization. Non-glucose sugars in the pulp decreases to values close to 0.0% DSB for the pretreatments performed in the most drastic conditions. Cellulose is also affected by the addition of sulfuric acid, but to a much lesser extent than hemicellulose. Acid addition causes the anhydrous glucose content to diminish constantly from 35.5% in experiment 17-S to 16.2%
in experiment 22-S. The fraction of ethanol/toluenesoluble lignin increases to 8% DSB and that of Klason lignin decreases to 14.1% from a value of 23.2% in experiment 15-S. The influence of the operating conditions on the yield and chemical composition for the hydrolytic pretreatment of almond shells, as well as the modeling of the kinetics of pentosan solubilization during the pretreatment using a severity parameter, has been presented and discussed in a previous work (Martı´nez et al., 1995). Description of the Softwood Pretreatment Using a Severity Parameter. The data discussed in the previous section suggest that it is possible to produce very similar changes in the chemical composition of the lignocellulosic substrate at multiple combinations of the three main operating conditions: temperature, time, and acid concentration. This behavior has been widely observed for other biomass species and enables the operation variables to be grouped into a severity parameter, which is then used as a single reaction ordinate (Overend and Chornet, 1987). This procedure has been previously applied to almond shells (Martı´nez et al., 1995). Its use in the hydrolytic pretreatment of the softwood mixture has been developed using the ROH severity parameter, which is defined by modeling the solubilization of hemicellulose, fif, during autohydrolysis and dilute-acid hydrolysis of lignocellulosics (Abatzoglou et al., 1992). The essential equations of the model are
ROH )
∫0t[min] exp
(
) (
)
T [°C] - Tref X - Xref γ-1 exp t dt ω λ (2)
fif ) 1 - exp(-KROH)
(3)
692 Ind. Eng. Chem. Res., Vol. 36, No. 3, 1997
Figure 2. Pretreatment of the softwood mixture in the continuous tubular reactor. Hemicellulose remaining in the pretrated pulp as a function of KROH: (autohydrolysis, O; dilute-acid hydrolysis, b; the continuous line is the model prediction.
where, according to Abatzoglou’s work (1992), ω ) constant dependent on the average activation energy of the reaction process, λ ) constant expressing the effect of the acid on the reaction rate, γ ) parameter that introduces a distribution of activation energies, K ) reaction rate in the reference conditions (Tref and Xref), and fif ) fraction of solubilized hemicellulose. These constants are dependent on the biomass considered and have to be obtained from experimental data. The use of the product KROH as a reaction ordinate furnishes an absolute scale that enables different biomass species to be compared. The constants were optimized using 100 °C as Tref and 0.0 g acid/kg of slurry as Xref. The optimum values of the constants for the softwood mixture were 0.067 min-0.36 for K, 60.04 °C for ω, 0.50 g of acid/kg of slurry for λ, and 0.36 for γ. The standard error, σn-1, was 0.042, which corresponds to a R2 of 0.94. The comparison between the experimental data and the model predictions is shown in Figure 2. Good agreement is observed, enabling KROH to be used as a reaction ordinate for the correlation of the pulp composition. The fraction of solubilized lignocellulosic for the two data sets reported in Tables 2 and 3 is shown in Figure 3 as a function of the pretreatment severity. The amount of water-soluble material produced during the hydrolytic pretreatments rises from a value of around 18% DSB at KROH ) 0.9 to 60% DSB at KROH ) 15 000, increasing continuously with severity. The same figure also shows the evolution of the Klason and the total lignin (Klason plus ethanol/ toluene-soluble lignin). There is a good agreement between the autohydrolysis and dilute-acid hydrolysis data. The content of Klason lignin decreases with severity from 25% DSB in the untreated softwood mixture to a value of 19% DSB at a KROH of 5. Klason lignin solubilization further decreases to 14% DSB at a KROH of 15 000, which represents around 56% of the Klason lignin in the untreated lignocellulosic. Simultaneously, the ethanol/toluene-soluble lignin continuously increases to a value close to 8% DSB at the highest severity studied. The total lignin content follows a trend similar to that of the Klason lignin fraction, decreasing from 27.4% DSB at KROH ) 0.9 to around 24.5% DSB at KROH ) 5, a value which remains practically constant at higher severity. From the content of anhydrous glucose in the treated pulp, cellulose does not appear to undergo a significant solubilization at severities below KROH ) 25. Beyond this point, glucose constantly diminishes from 37% to 16% DSB at the highest severity considered here, which represents around 42% of the glucose contained in the untreated lignocellulosic. Thus, cellulose is depolymer-
Figure 3. Pretreatment of the softwood mixture in the continuous tubular reactor. Solubilization and chemical composition of the insoluble fraction as a function of KROH. Solubilization: autohydrolysis, O; dilute-acid hydrolysis, b. Anhydrous glucose: autohydrolysis, ]; dilute-acid hydrolysis, [. Total lignin: autohydrolysis, 4; dilute-acid hydrolysis, 2. Klason lignin: autohydrolysis, 3; dilute-acid hydrolysis, 1. The dashed lines only indicate trends.
ized simultaneously with hemicellulose, but at a significantly lower rate. Cellulose Average Degree of Polymerization. The viscosity-average degree of polymerization, DPv, of the R-cellulose remaining in the pulp after hydrolytic pretreatment is shown in Tables 2 and 3 for the softwood mixture and in Tables 4 and 5 for the almond shells. The DPv of the cellulose in the softwood mixture gradually decreases as the pretreatment is performed at higher severity. Figure 4 shows this evolution as a function of KROH. The DPv decreases from a value of 700 for the untreated softwood mixture to around 200 for the samples pretreated at a KROH of 10. This value of DPv corresponds to the level off degree of polymerization (LODP) for this substrate since further pretreatment in much more severe conditions (higher reaction temperature, time, and acid concentration) does not produce any significant reduction in the R-cellulose DPv. At a KROH higher than 10 000, the DPv is still 180. Figure 4 also shows the evolution of the DPv for the R-cellulose fraction in almond shells, an agriculture waste with an initial R-cellulose DPv also close to 700, pretreated in the same tubular reactor in operating conditions similar to those for the pretreatment of the softwood mixture. The DPv of the R-cellulose fraction of both substrates behave in a very similar way when the pretreatment severity is varied. The results in Figures 3 and 4 show that the KROH parameter may be used not only as a description of the kinetics of hemicellulose solubilization but also as a common reaction ordinate to group the results of different biomass species pretreated in similar operating conditions. However, using KROH does not always enable several data sets to be grouped into a single trend line. Figure
Ind. Eng. Chem. Res., Vol. 36, No. 3, 1997 693 Table 4. Pretreatment of Almond Shells by Autohydrolysis and Dilute-Acid Hydrolysis in the Continuous Tubular Reactor (First Configuration)a experiment number experimental conditions temperature (°C) time (min) acid load (g of acid/kg of slurry) log(KROH) R-cellulose DPv a
6-A
7-A
8-A
9-A
10-A
11-A
12-A
13-A
14-A
15-A
16-A
215 1.0 0.0 0.05 540
210 1.2 0.0 0.01 580
211 1.2 0.0 0.03 560
210 1.1 0.4 0.07 550
209 1.1 0.7 0.13 540
211 1.1 0.7 0.17 480
210 1.0 0.8 0.13 460
210 1.0 1.3 0.27 410
209 1.0 1.5 0.30 330
211 1.0 2.2 0.50 260
207 1.0 2.2 0.42 310
Operating conditions and DPv of R-cellulose in the pretreated pulp.
Table 5. Pretreatment of Almond Shells by Autohydrolysis in the Batch Stirred Reactora experiment number experimental conditions temperature (°C) time at temperature (min) average heating rate (°C/min) cooling rate (°C/min) log(KROH) R-cellulose DPv a
18-A
19-A
20-A
21-A
22-A
23-A
24-A
26-A
27-A
28-A
180 3.0 9.1 31 -0.18 360
180 5.0 8.2 39 -0.19 420
180 9.0 7.8 31 0.04 330
180 10.0 9.7 38 0.14 360
180 17.0 6.0 26 0.15 330
180 20.0 5.0 26 0.24 310
180 38.0 7.0 22 0.49 310
200 3.0 10.3 35 0.19 350
200 8.0 8.8 17 0.32 280
200 44.0 8.8 20 0.72 250
Operating conditions and DPv of R-cellulose in the pretreated pulp.
Figure 4. Pretreatment of the softwood mixture and the almond shells in the continuous tubular reactor. DPv of the R-cellulose in the pulp as a function of KROH. Almond shells: autohydrolysis, 0; dilute-acid hydrolysis, 9. Softwood mixture: autohydrolysis, O; dilute-acid hydrolysis, b. The dashed line only indicates the trend.
Figure 5. Pretreatment of the almond shells in the continuous tubular reactor and the batch stirred reactor. DPv of the R-cellulose fraction present in the pulp as a function of KROH. Tubular: autohydrolysis, 0; dilute-acid hydrolysis, 9. Batch: autohydrolysis, O. The dashed lines only indicate trends.
5 shows the evolution for the DPv of the R-cellulose fraction that remains in the almond shells after hydrolytic pretreatment in the continuous plug flow reactor and in the batch stirred autoclave. There are two welldefined trends which depend on the reactor unit used for the pretreatment. This is consistent with the concept of using KROH as a reaction ordinate, since this parameter measures the severity of the process from the point of view of the kinetics of hemicellulose solubili-
zation. From eqs 2 and 3, any combination of temperature, time, and acid concentration that results in a single value of KROH will provide a single value of hemicellulose solubilization. Considering that the average reaction rates for hemicellulose and cellulose depolymerization are obviously different, then two sets of temperatures, times, and acid concentrations that result in the same value of KROH, and hence produce the same extent of hemicellulose solubilization, will not depolymerize cellulose to the same degree. The larger the difference between the rate constants (i.e., k0 and Ea) that describe the hydrolytic process for hemicellulose and cellulose, the larger the difference between the degrees of polymerization for each set of operating conditions will tend to be. So the distinct trends observed in Figure 5 for the DPv of R-cellulose samples obtained in the stirred autoclave and the plug flow reactor are explained by the dissimilar operating conditions used in both data sets: (a) Stirred batch unit: 180 or 200 °C, 1-68 min, autohydrolysis. (b) Continuous plug flow tubular unit: 180-215 °C, around 1 min; from 0 to 2.2 g of acid/kg of slurry. Consequently, besides describing the kinetics of hemicellulose solubilization, KROH may be used to group other properties of different lignocellulosic pulps if the range of operating conditions used for each substrate lies within the same region. When dissimilar operating conditions are used, KROH is still capable of modeling the kinetics of hemicellulose removal for a specific substrate and also of providing valuable information about the performance of the process for each range of operating conditions. Data in Figure 5 show that for the same extent of hemicellulose solubilization (same KROH value), the reduction in the DPv of cellulose is less important when the pretreatment is performed at high temperatures and short residence times (tubular reactor). On the contrary, low temperatures and high residence time (stirred batch unit) cause an extensive depolymerization of the cellulose, even at low severity. This behavior is not restricted to almond shells but also seems to be the case for most biomass species. Heitz and co-workers (1987) studied the depolymerization of cellulose during the pretreatment of eucalyptus under autohydrolysis conditions and described the evolution
694 Ind. Eng. Chem. Res., Vol. 36, No. 3, 1997
Figure 7. Enzymatic hydrolysis of the pretreated softwood mixture and the almond shells. Glucose yield during enzymatic saccharification as a function of the DPv of the R-cellulose present in the pretreated pulp. Almond shells: untreated, 3; autohydrolysis, 0; dilute-acid hydrolysis, 9. Softwood sawdust: untreated, 4; autohydrolysis, O; dilute-acid hydrolysis, b. The dashed lines only indicate trends.
Figure 6. Evolution of the cellulose DPv during autohydrolysis at constant temperature for eucalyptus, almond shells, and the softwood mixture. Table 6. Cellulose Depolymerization during Autohydrolysis Pretreatmenta species eucalyptusb almond shells softwood mixture
DPv0
A (s-1)
E (kJ‚mol-1)
R2
950 700 700
3.4 × 3.0 × 103 2.5 × 103
78.8 72.6 74.3
0.91 0.88
104
a Values of the parameters in Basedow’s model. bFrom Heitz et al., 1987.
of the DPv using the correlation proposed by Basedow (Ederer et al., 1980) for soluble polymers:
DPv )
DPv0 (1 + (DPv0)aakt)1/a
(-E RT )
k ) A exp
(4)
(5)
where a ) 2/3 for hydrolysis, t ) time (min), A ) frequency factor (s-1), E ) activation energy (kJ‚mol-1), DPv ) viscosity-average degree of polymerization of the pretreated sample, and DPv0 ) viscosity-average degree of polymerization of the untreated sample. Figure 6 compares the effect of the autohydrolysis temperature on the DPv of the cellulose fraction for eucalyptus as a function of the reaction time. Calculations were made at two temperatures, 180 and 210 °C, using the values of A and E obtained by Heitz and coworkers (1987), which are listed in Table 6. Although it is clear that the rate of depolymerization rises with temperature, the advantage of using high temperatures and short residence times can be seen when the comparison is performed on the basis of a temperaturetime combination that produces an equal amount of hemicellulose solubilization. Thus, autohydrolysis at 210 °C reduces the DPv from 950 to 570 after 1 min, but a treatment that reaches an equivalent degree of
hemicellulose solubilization at 180 °C (8 min) reduces the DPv to 370 units. For eucalyptus, the equivalence among pretreatment conditions for hemicellulose solubilization has been calculated using the R0 parameter (Overend and Chornet, 1987), since no data were available to apply KROH to this species. Basedow’s model was subsequently applied to the modeling of R-cellulose depolymerization during the autohydrolysis of almond shells and the softwood mixture. Table 6 lists the activation energies and the frequency factors calculated from the experimental values of DPv for each species, and Figure 6 compares depolymerization at two combinations of temperature and reaction time that produce the same extent of hemicellulose depolymerization of each substrate. The results for the three match the trend deduced from the experimental results for almond shells, discussed in Figure 5. This is of maximum importance for the fractionation of biomass into its constitutive polymers, since fractionation aims to separate the cellulose, hemicellulose, and lignin with a high yield of soluble hemicellulose and a minimum of depolymerized cellulose. High temperatures and short residence times not only promote hemicellulose solubilization rather than cellulose depolymerization but also give higher yields of recovered hemicelluloses since the reactions involved in the degradation of the soluble hemicellulose in the aqueous phase are actually slower than the solubilization process under these conditions (Converse et al., 1989). High temperatures and short residence times do, however, have some physical limits: 1. There is a maximum temperature of 250 °C, since hemicellulose pyrolysis begins to predominate over hydrolysis at higher values. 2. The shortest pretreatment time that may be used is directly related to the size of the lignocellulosic particles. For reactor systems using commercial-size chips, like in steam-explosion processes, steaming times under 2 min may result in an incomplete reaction at the center of the chips (Brownell et al., 1986), and so this is the shortest reaction time adopted. When the lignocellulosics are pretreated as ground material in aqueous slurries, reaction times as low as 0.5 min can be successfully used (Converse et al., 1989). Susceptibility to Enzymatic Hydrolysis. Glucose yield after enzymatic saccharification of the pretreated substrates, expressed as percent of the potential glucose in the pretreated substrate, is shown in Figure 7 for both the softwood mixture and the almond shells as a function of the degree of polymerization of the R-cel-
Ind. Eng. Chem. Res., Vol. 36, No. 3, 1997 695
lulose fraction. Two main conclusions can be drawn from these results: 1. Softwood cellulose is extremely resistant to enzymatic hydrolysis. Only after a very severe pretreatment, which results in an R-cellulose DPv lower than 250, there is a net increase in the saccharification yield. However, the maximum yield obtained in this study barely surpasses 44% of the potential glucose present in the pretreated pulp, and only after reaching the LODP of the R-cellulose and solubilizing nearly 58% of the potential glucose of the substrate during the hydrolytic pretreatment. 2. Almond shell cellulose is more susceptible to enzymatic hydrolysis and behaves in this sense like a hardwood substrate. In fact, Montane´ et al. (1993) observed that results of xylan solubilization and recovery were in concordance with those reported for Populus tremuloides pretreated in a Stake II system (Heitz et al., 1991). There is a continuous increase in the glucose yield during enzymatic hydrolysis as the pretreatment depolymerizes the cellulose to lower values of DPv, reaching a yield of 97% of the potential glucose in the substrate for the sample pretreated at the maximum severity studied (DPv of 260). Several factors have been suggested to try to explain way softwood species exhibit such an extreme resistance to enzymatic hydrolysis. It has been suggested that the reduction in the cellulose DPv causes an increase in the number of chain ends available to the action of the exoglucanase in the cellulose complex, thus producing an increase in the reaction rate and glucose yield during enzymatic hydrolysis (Converse, 1993). Although the relation between the reduction in DPv and the improvement in glucose yield during enzymatic hydrolysis is well documented for hardwood species (Puls et al., 1985; Heitz et al., 1991) and has been verified for almond shells in this study, results for the softwood mixture indicate that other factors have to be taken into account if this behavior is to be explained. The improvement in the enzymatic digestibility of cellulose in pretreated wood substrates has been related to the removal of the hemicellulose fraction (Grohmann et al., 1985), which according to Grethlein (1991) results in an increase in the pore volume and the specific surface area of the cellulose that is accessible to the enzyme molecules. The solubilization of cellulose during the pretreatment must also help to improve accessibility as digestibility continues to increase after all the hemicellulose has been removed (Wong et al., 1988; Clark et al., 1989). So, Figure 8 shows the yield during enzymatic saccharification as a function of the amount of carbohydrates solubilized during the pretreatment for both lignocellulosic species. Although it appears that there is a direct relationship between the removal of cell wall carbohydrates and the increase in the digestibility of the remaining cellulose, there is still a significant difference between the behavior of the softwood mixture and almond shells. Such a discrepancy may be attributed to the fact that the cellulose matrix is not open to the same extent during hemicellulose removal in softwoods as it is in hardwoods, resulting in the development of smaller pores in softwoods and thus in an incomplete conversion of cellulose due to insufficient accessibility to the enzyme molecules (Grethlein et al., 1984). A different distribution in the lignocellulosic matrix of the lignin depolymerized during the pretreatment of softwood and almond shells may be another
Figure 8. Enzymatic hydrolysis of the pretreated softwood mixture and the almond shells. Glucose yield during enzymatic saccharification as a function of the carbohydrates solubilized during the pretreatment. Almond shells: untreated, 3; autohydrolysis, 0; dilute-acid hydrolysis, 9. Softwood sawdust: untreated, 4; autohydrolysis, O; dilute-acid hydrolysis, b. The dashed lines only indicate trends.
factor interfering with the action of the enzymatic complex in the case of softwoods. Conclusions Autohydrolysis and dilute-acid hydrolysis were studied as pretreatment methods to increase the reactivity of cellulose in softwood and almond shells to enzymatic hydrolysis. Results obtained from two reactor systems with very different temperature-time combinations, an isothermal plug-flow reactor and a nonisothermal stirredbatch autoclave, can be unified and modeled using a severity parameter, KROH based on the kinetics of hemicellulose sugar solubilization during the hydrolytic pretreatment. This parameter is a valuable tool for correlating pulp properties such as the chemical composition and the DPv of the R-cellulose remaining in the pulp. Raising the pretreatment severity causes the DPv of the softwood cellulose to decrease from a value of 700 for the untreated substrate to around 200 at a KROH of 10, which corresponds to the leveling-off degree of polymerization as no significant variation in DPv occurs above this severity. R-Cellulose in the softwood mixture and almond shells is depolymerized to an equivalent extent when the pretreatment is carried out under the same operating conditions in the continuous tubular reactor. By contrast, differences can be observed in the extent of depolymerization for almond shells pretreated at the same severity range under very dissimilar timetemperature combinations. When the pretreatment conditions are examined on the basis of a temperaturetime association that produces an equal extent of hemicellulose solubilization, the use of high temperature and short residence time accelerates hemicellulose solubilization over cellulose depolymerization, thus resulting in a less degraded cellulose. The yield during enzymatic saccharification of the pretreated substrate is different for the two lignocellulosics compared in this study. The origins of this discrepancy are not clear, since it appears that the cellulose in both substrates is depolymerized to a similar extent during the pretreatment. Removing hemicellulose sugars improves the enzymatic hydrolysis yield for both biomass species, although this effect is greater for almond shells than for the softwood mixture at the same extent of carbohydrate removal. A more thorough study should be carried out to determine more precisely the nature of the differences
696 Ind. Eng. Chem. Res., Vol. 36, No. 3, 1997
in the saccharification yield for the two pretreated substrates. Acknowledgment The authors are indebted to CICYT and Generalitat de Catalunya (Catalan Regional Government, Spain) for financial support (Project No. QFN95-4720). Notation A ) frequency factor (s-1) CED ) cupriethylenediamine hydroxide DPv ) viscosity-average degree of polymerization of the R-cellulose fraction % DSB ) percent dry solid basis E ) activation energy (kJ‚mol-1) fif ) fraction of solubilized hemicellulose IU/mL ) international units of enzymatic activity (mol of glucose produced/min‚mL) K ) reaction rate at the reference conditions (min-γ) LODP ) leveling-off degree of polymerization ROH ) severity factor (Abatzoglou et al., 1992) t ) time (min) T ) temperature (°C) Tref ) reference temperature (°C) X ) acid catalyst concentration (g of acid/kg of slurry) Xref ) acid catalyst reference concentration (g of acid/kg of slurry) Greek Letters λ ) parameter expressing the influence of the acid catalyst ω ) parameter related to the average activation energy of the process, expressing the influence of temperature γ ) parameter introducing a distribution of activation energies [η] ) limiting viscosity number (mL‚g-1)
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Received for review January 23, 1996 Revised manuscript received November 11, 1996 Accepted November 14, 1996X IE960048E
X Abstract published in Advance ACS Abstracts, January 1, 1997.
