Kinetics Study of Enzymatic Hydrolysis of Paulownia by ... - Springer Link

Biotechnology and Bioprocess Engineering 20: 242-248 (2015) DOI 10.1007/s12257-014-0490-x

RESEARCH PAPER

Kinetics Study of Enzymatic Hydrolysis of Paulownia by Dilute Acid, Alkali, and Ultrasonic-assisted Alkali Pretreatments Xiao-kun Ye and Yuancai Chen

Received: 11 July 2014 / Revised: 3 November 2014 / Accepted: 8 November 2014 © The Korean Society for Biotechnology and Bioengineering and Springer 2015

Abstract Paulownia, a fast-growing and high-fiber plant (cellulose: 41.66% and hemicellulose: 19.61%), has the potential to serve as an interesting source for production of bioethanol. The aim of this paper is to study and compare the kinetics of enzymatic hydrolysis of Paulownia pretreated by dilute acid (DA), alkali (AL) and ultrasonic-assisted alkali (UA). The enzymatic hydrolysis was performed at 50°C, atmospheric pressure with 40 FPU/g-cellulose cellulase and 80 CBU/g-cellulose cellobiase. The hydrolysis process can be successfully described by the Michaelis-Menten model under the three pretreatment conditions. Due to the high removal of lignin and increased porosity of the substrate, UA pretreatment is proved to be the most effective method in improving enzymatic saccharification, followed by DA pretreatment and alkali (AL) pretreatment. Inhibition constant KI of all experiments (DA: 2.16 g/L, AL: 3.12 g/L and UA: 1.83 g/L) suggests that glucose has a strong inhibition for enzymatic hydrolysis, for lower KI is proportional to higher inhibition performance. The experimental date fits well with kinetics model. This indicates that the model is suitable for performance monitoring, conditions optimization and process control at full-scale studies. Keywords: Paulownia, enzymatic hydrolysis, kinetics, dilute acid, ultrasonic-assisted alkali, pretreatment

Xiao-kun Ye College of Environment and Energy, South China University of Technology, Guangzhou 510-006, China Yuancai Chen* College of Environment and Energy, South China University of Technology, Guangzhou 510-006, China State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510-640, China Tel: +86-136-724-580-60; Fax: +203-9380-599 E-mail: [email protected]

1. Introduction The finiteness of fossil fuels leads to the wide study in bioethanol nowadays. As a renewable energy, biofuel can help to reduce greenhouse gas emissions. Since it consumes a large amount of cereal crops and creates a series of economic problems, the first generation bioethanol production cannot really achieve sustainable development. It is proposed that lignocellulose shall replace crops as raw material to produce bioethanol and other chemicals [1-5]. Various lignocellulose-rich plants, such as wheat straw [1], rice straw, sugar cane bagasse [2], water hyacinth [3] and sorghum [4] have been studied for the second generation bioethanol production. Although these wastes used for bioethanol can speed up the material circulation, the content of lignocellulose, especially cellulose, is low, which increases the using of chemicals in treating process. This study chooses Paulownia tomentosa, a plant grows widely in northeast, east, central and northwest China. As a quickgrowing hardwood plant, it takes about 5 years to grow to 30 ~ 40 cm in diameter at breast height. Our previous experiments revealed that Paulownia mainly contains three separate constituents: cellulose (41.66%), hemicellulose (19.61%) and lignin (20.46%) [5,6]. The feature of fast growth and low lignin content suggests that it can be a better source for production of fuel alcohol than other plants. Cellulose is surrounded tightly by lignin and hemicelluloses. Complexity of the structure of the lignin hinders the accessibility of enzyme to cellulosic fibers. Therefore pretreatment is required to alter the structure and chemical composition of lignocellulosic biomass. A series of pretreatment techniques including physical (comminution, hydrothermolysis), chemical (acid, alkali, solvents, ozone), physicochemical (steam explosion, ammonia fiber explosion) and biological methods had been developed for the

Kinetics Study of Enzymatic Hydrolysis of Paulownia by Dilute Acid, Alkali and Ultrasonic-assisted Alkali Pretreatments

dissolution of hemicellulose and removal of lignin to improve enzymatic hydrolysis efficiency [7-9]. Aguiar [10] pretreated sugarcane bagasse using a physical (steamexploded) method at 210°C in high pressure for 4 min, acquired higher reducing sugar yield. However, pressurization and sudden depressurization need a huge amount of energy. Biological method, an environmental friendly and resource saving way, was presented to remove lignin before enzymatic hydrolysis. Previous studies suggest that whiterot fungi (notably Phanerochaete chrysosporium) could degrade lignin with the help of manganese peroxidase, lignin peroxidase and laccase. It was also reported that bacteria also have the ability to degrade lignin [11,12]. However, strict microbial growing conditions, longer processing cycle and complex mechanism of degradation impede the wide application of biological method. Chemical pretreatment can achieve high efficiency of lignin removal and enzymatic hydrolysis. In particular, dilute acid (DA) and alkali (AL) pretreatments have been widely applied, because their experimental conditions are mild, efficient and inexpensive [13]. Several previous experiments were carried out to investigate the effect on enzymatic hydrolysis using dilute acid (DA) pretreatment. Chen [14] found that dilute sulfuric acid pretreatment can effectively convert hemicellulose into monosaccharide (xylose, arabinose, galactose and mannose) and soluble oligomers. Grohmann [15] reported the degradation of wheat straw and aspen wood using dilute sulfuric acid pretreatment at 140°C for 1 h and observed that about 80% degradation of xylan. Ayse Avciet [16] investigated the pretreatment of corn stover using dilute phosphoric acid. About 91.4% of the xylose and 85% of enzymatic hydrolysis efficiency were observed when it was pretreated by 0.5% (v/v) H3PO4 at 180°C for 15 min. Most of studies reveal that most hemicellulose are solubilized when experiments are performed in presence of as much as acid, some glucose is degraded to hydroxymethyl furfural (HMF), formic acid, acetic acid and furfural. The existence of these intermediates reduced sugar fermentation efficiency by inhibiting yeast growth [17,18]. Therefore, pretreatment with DA was proposed as detoxification strategies [18]. Meanwhile, many findings suggest that alkali has the ability to disrupt lignin structure and obtain high enzymatic digestibility efficiency. For instance, the removal of lignin and hemicellulose was 74% and 55% with the 2.5% NaOHtreatment at 105°C for 10 min [19]. The removal of lignin and decomposition of glucan achieved 78% and 98%, respectively, when Sugarcane bagasse was conducted in 0.25 M NaOH solution at 80°C for 3 h [20]. Using ultrasonic as pretreatment method was rarely reported. However, due to its low cost and high glucose yield, it is gaining popularity when it is used in tandem with efficient chemical

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methods. The kinetics of enzymatic hydrolysis plays an important role in mechanism of enzymatic hydrolysis and process control. Cellulose hydrolysis process is influenced by many factors, such as materials, pretreatment methods, enzyme dosages, pH and temperature. This leads to producing different kinetic parameters under different condition [21]. In addition, other factors about enzymes such as diffusion, inactivation, transformation, inhibition of enzymatic hydrolysis process and the mutual influence between these factors also cause different description of the hydrolysis process for different study. Therefore, it is of great significance to study Paulownia kinetics under certain conditions. The objective of this paper is to assess and compare three different pretreatment methods: (dilute sulfuric acid (DA), alkali (AL), ultrasonic-assisted alkali (UA)) of Paulownia by building kinetics model of enzymatic hydrolysis for maximizing recovery of valuable materials from Paulownia under mild operating conditions.

2. Materials and Methods 2.1. Materials Paulownia samples were obtained from a suburb of Shanxi province, China. They were milled and a fraction (< 0.3 mm) was separated using a 60 ~ 80 mesh sieve by a high-speed universal grinder (FW100, Tianjin Taisite Instrument Co. LTD, China). Samples were stored in plastic bags at room temperature in the lab prior to experiment. 2.2. Dilute Acid (DA), Alkali (AL), and Ultrasonicassisted Alkali (UA) pretreatment DA pretreatment was performed in a beaker and kept at 140°C in thermostatic oil bath pot (HH-S) for 30 min using Paulownia with 50 mL of 1.2% H2SO4. For AL pretreatment, washed Paulownia was pretreated in 100 mL of 10% NaOH for 15 min at 80°C with a solid-liquid ratio of 10% (w/v). UA pretreatment was performed in ultrasonic CNC cleaning machine (KQ-300DE, Kunshan ultrasonic numerical control Co. LTD) with ultrasonic power of 60 W and 100 mL of 10% NaOH for 15 min at 80°C. All of experiments were performed under optimal conditions obtained from our previous study. After pretreatment, liquid fractions were collected and various kinds of monosaccharide (glucose, xylose, galactose and arabinose) and lignin concentration were measured using a DIONEXI CS300 chromatography system. The solid fractions were collected and washed with distilled water several times until neutral pH was achieved. The samples then were dried at 55°C for 2 h by digital display drum wind drying oven (GZX-9076) and weighed. Finally,

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Biotechnology and Bioprocess Engineering 20: 242-248 (2015)

the substrates were sealed and kept at 4°C. 2.3. Enzymatic hydrolysis of cellulose Solid fractions of pretreated Paulownia were further hydrolyzed by mixture of commercial cellulase (40 FPU (filter paper unit)/g-cellulose) and cellobiase (80 CBU (cellobiase unit)/g-cellulose), which were generated from Trichoderma reesei ATCC 26921 and Aspelgillus niger (Sigma-Alorich), respectively. To reduce the influence of enzyme limitation on sugar yields, excessive cellulose and cellobiase were added. Sodium azide (0.3% w/v) was added into the mixture to prevent microbial contamination. All batch reactors were performed with 50 mL of 0.1 M sodium acetate buffer (pH = 4.8) in Erlenmeyer flask, and carried out with 150 rpm of shaking for 150 h at 50°C on a thermostatic water bath shaker. 1 mL solution were sampled and determined at every 12-hour during the hydrolysis of fibrous substrates. When enzymatic hydrolysis was completed, the hydrolysate was heated for 5 min in a boiling water batch, centrifuged at 7,000 rpm for 15 min to ensure inactivation of enzyme and system stability. The quantification of reducing sugar concentration was conducted by spectrophotometric analysis using the 3,5-dinitrosalicylic acid (DNS method, ACROS, New Jersey, USA) [22] and described by glucose equivalent. Enzymatic hydrolysis efficiency (EHE) was evaluated using the reducing sugar yield based on raw biomass. All of experiments were conducted in duplicate. 2.4. Model description Cellulose hydrolysis is a solid and liquid interphase reaction between soluble cellulose enzyme and undissolved solid cellulose molecules. Michaelis-Menten model was used to simulate the cellulose hydrolysis in the study [23]. Three assumptions were made in the experiment: (1) Cellulose hydrolysis reaction is a single pseudo-homogeneous system; (2) The cellulose of pretreated Paulownia is composed of enzymatic hydrolysis fraction and un-enzymatic hydrolysis one; and, (3) Enzymatic hydrolysis reaction is affected by competitive inhibition of glucose products. Thus, the reaction rate, dC ------- (g/(L/h)), can be expressed by dt the Michaelis–Menten equation as follows: RmaxCs dC ------- = -------------------------------------dt C⎞+C KM⎛1 + -----s ⎝ KI ⎠

inhibition. Since the kinetics experiments were conducted with batch bottles of pretreated Paulownia, the Michaelis-Menten equation was modified: Rmax(Cs0 – 0.9C) dC ------- = -------------------------------------------------------dt C⎞ ⎛ KM 1 + ------ + (Cs0 – 0.9C) ⎝ KI ⎠

(2)

Where Cs0 is the initial cellulose concentration of enzymatic hydrolysis of pretreated Paulownia (g/L), 0.9 indicates 90% starch transforms into reducing sugar. Other parameters are the same as those in Eq. (1).

3. Result and Discussion 3.1. Characterization of prehydrolysate The characteristic of Paulownias is described in detail elsewhere [6]. It mainly consists of three separate constituents: cellulose (41.66%), hemicelluloses (19.61%) and lignin (20.46%). Glucan, acid-insoluble lignin, xylan, arabinan and galactan are 41.66, 18.89, 17.56, 0.86, and 1.27%, respectively. It can be seen that Paulownia’s composition is comparable with the Messmate, the widely accepted good fiber material [24]. The production of bioethanol needs three processes. Firstly, remove lignin and dissolve hemicellulose from Paulownia using pretreatment techniques. Secondly, hydrolyzes cellulose into reducing sugars by enzyme. Thirdly, convert fermentable sugars to bioethanol by yeast or bacteria [1]. As shown in Fig. 1, three pretreatment methods change the composition of prehydrolysate (liquid fractions) considerably. The performance of UA leads to a little higher glucose (major hydrolysis products of cellulose) product

(1)

Where C is the reducing sugar concentration (g/L), t is the reaction time (h), Rmax is the maximum reaction velocity (g/L·h), KM is the Michaelis-Menten constant (g/L), and Cs is the cellulose concentration of enzymatic hydrolysis. KI is inhibition constant (g/L) and a larger KI indicates that the culture is less sensitive to substrate

Fig. 1. Four main monosaccharide and lignin concentration of hydrolasate after dilute acid (DA), alkali (AL), ultrasonic-assisted alkali (UA) pretreatment.


yield (111.56 mg/g) in prehydrolysate after pretreatment, compared with AL (89.50 mg/g) and DA (89.32 mg/g), which suggests higher cellulose lose during the UA. Xylose (major hydrolysis products of hemicellulose) concentration pretreated by DA is 156.94 mg/g, far more than other two treat methods (AL: 95.32 mg/L UA: 120.08 mg/L). In contrast, regardless of high delignification effect AL pretreatment is not able to remove xylan of hemicellulose entirely [25]. The result reveals that DA selectively removes hemicelluloses of Paulownia without causing too much sugar losses. For lignin, although its amount was almost equivalent to AL and UA the higher delignification rate is observed as the result of UA treatment (165.96 mg/g), which indicates that ultrasonic can assist alkali pretreatment prominently. Apparently, delignification effect of DA is worse than AL. As for arabinose and galactose yield, they do not greatly differ with each other for all samples due to their original low contents. 3.2. Enzymatic hydrolysis The effects of three different pretreatment methods on the following enzymatic hydrolysis process were studied. Solid fractions of pretreated Paulownia were hydrolyzed by excess enzyme to fermentable sugars. Fig. 2 shows that reducing sugar concentrations increase gradually with time, the concentration of reducing sugar in liquid fraction of pretreated Paulownia (DA, AL and UA) are 349.45, 414.48, and 367.60 mg/g, respectively, after 120 h of enzymatic saccharification, and the corresponding enzymatic hydrolysis efficiencies (EHE) are 89.3, 88.5 and 91.7%, respectively after 120 h. UA leads to maximum

production of reducing sugar and enzymatic hydrolysis efficiency which can be explained by the joint pretreatment effect between alkali and ultrasonic. AL treatment firstly break the ester bonds between lignin and xylan to increase the porosity of the biomass [26]. Then, swelling effect of AL treatment may also lead to an increase in the surface area. Moreover, ultrasonic irradiation to liquid facilitates mass transfer of the solute and solid fractions in liquids. Some researchers demonstrate that ultrasonic irradiation at 23 ~ 25 kHz can increase the porosity of cellulose fiber and cleave α-O-4 or β-O-4 of lignin [27,28]. Therefore, the porosity of cellulose fiber and cleavage of lignin provide larger contact opportunity between pretreated Paulownia and enzyme. Additionally, UA pretreatment can effectively partially hydrolyze lignin and hemicelluloses to watersoluble monomers and oligomers, while crystallinity of cellulose and the degrees of polymerization are destroyed. This improvise its susceptibility to enzymatic hydrolysis. Similar results were also reported by Gupta and Nazhad [29,30]. Disaccharide was not found in the hydrolysate after enzymatic hydrolysis. It can be inferred that the enzyme complex used for the hydrolysis has high activities of exo-β-1,4-glucanases, endo-β-1,4-glucanasesand and β1,4-glucosidases [31]. 3.3. Determination of model parameter Rmax , KM , and KI The Michaelis-Menten kinetics model was applied to determine the kinetic parameters on the basis of the amount of products generated during enzymatic hydrolysis. Because the reducing sugar concentration is approximately equal to 0 ( C ≈ 0 ) in the initial stage of Paulownia hydrolysis process, so Eq. (2) can be simplified into: KM 1 1 1 ⎞ ⎛-------------= ---------------- + ---------⎝ dC/dt ⎠ t → 0 Rmax Cs0 Rmax

Fig. 2. Enzyme hydrolysis of three different pretreated Paulownia at 50°C for 150 h. (Dilute acid pretreatment conditions: 30 min, T = 140°C, 1.2% H2SO4. Alkali pretreatment conditions: 15 min, T = 80°C, 10% NaOH. Ultrasonic-assisted pretreatment conditions: 15 min, T = 80°C, 10% NaOH, 60 W) Experimental values are plotted as makers and the curves are the modellisation of Eq. (2).

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(3)

The relationship between 1/(dC/dt)t → 0 and 1/Cs0 was investigated, initial velocity was determined based on reducing sugar generated on average in enzyme hydrolysis processes for 30 min. As shown in Fig. 3, good linear correlation exist between 1/(dC/dt)t → 0 and 1/Cs0 for three pretreatment processes. This validates the applicability of Eq. (3) to the present substrate system under the condition studied. From the intercept and slope of the equation in Fig. 3, Rmax values of DA, AL and UA are 6.52,7.38 and 27.26 g/L, KM values are 13.02, 15.94 and 51.03 g/L, respectively. To determine glucose inhibition constant KI, the following linearized form of Eq. (2) is used: KMI 1 1 ⎞ 1⎛-------------= -------------- + --------⎝ dC/dt ⎠ t → 0 Rmax Cs Rmax and

(4)

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Fig. 3. Relationship between 1/(dC/dt)t→0 and 1/Cs0 during enzymatic hydrolysis.

KMI = KM(1 + C0/KI)

(5)

Glucose was added to the medium at a required sugar concentration before enzymatic hydrolysis. The relationship between the reaction rate (dC/dt, g/(L/h)) and the substrate concentration (Cs, g/L) was investigated. Inhibition constant (KI, g/L) was determined by different glucose concentrations. The experimental results of the enzymatic hydrolysis of different pretreated Paulownia at different glucose concentrations are showed in Fig. 4. It shows that the reaction velocity drops with the increases of glucose concentration during enzymatic hydrolysis. It suggests that enzymatic hydrolysis velocity of substrate is inhibited by glucose and the level of inhibition is improved with the increases of glucose concentration. Meanwhile, The KI of DA, AL, and UA pretreatment determinated from Eq. (5) are 2.16, 3.12, and 1.82 g/L, respectively. 3.4. Analysis of the kinetics model parameters The resulting Rmax and KM reflect not only the affinity between zymoprotein and solid fractions but also the dependence on the nature of substrate and experimental conditions. A small KM value and a large Rmax value indictate the strong affinity of zymoprotein and solid fractions. The kinetics parameters obtained from the Michaelis-Menten model of enzymatic hydrolysis of cellulosic materials by DA, AL and UA were assessed and the results are shown in Table 1. The high Rmax and low KM value are achieved with UA. The combination of alkali and ultrasonic when compared with the AL pretreatment, suggesting that sonication is useful in accelerating the enzymatic hydrolysis of cellulose. Although alkali treatment process have a great effect as the role in swelling of cellulose and removal of lignin, UA can significantly enhance hydrolysis efficiency of Paulownia as expected. Cavitation phenomenon pro-

Fig. 4. Relationship between 1/(dC/dt)t→0 and 1/Cs0 during enzymatic hydrolysis of different pretreated Paulownia with different glucose concentration. The dates presented are averages of three experiments. Symbols: glucose concentration (▲) 40 g/L (●) 20 g/L (■ ) 0 g/L.

duced by ultrasonic during UA process, causes the vibrations of the micro-bubble in the medium and a series of friction and collision between the cellulose molecules. It significantly increases the porosity of fiber and reduces the degree of crystallinity of fiber structure. A large Rmax value (16.80 mg/(L·h)) is obtained from orange peel waste hydro-


Table 1. Kinetics parameters of Paulownia hydrolysis from regression analysis of experimental results using Eq. (2) Kinetic parameters Pretreatment method

Rmax (g/(L/h)) Dilute acid (DA) 6.52 Alkali (AL) 7.38 Ultrasonic-assisted alkali (UA) 7.89

KM (g/L) 13.02 15.94 14.78

KI (g/L) 2.16 3.12 1.82

lysis after the intense ultrasonic treatment. This indicates that ultrasonic promotes enzymatic hydrolysis rate [32]. Compared to DA pretreatment, 12% improvement in Rmax and 18% improvement in KM during AL pretreatment are obtained. It is difficult to assess which pretreatment method is the best for enzymatic hydrolysis from the point of parameters of Rmax and KM. But from the perspective of experimental date, DA pretreatment has a better effect on enzymatic hydrolysis than alkali impregnation. Because DA pretreatment has a great effect on the degradation of hemicellulose (83.11%) and good removal of lignin (65.30%), while raw materials have hemicellulose of 19.61% and lignin of 20.46%, resulting in decreasing the crystallinity of the Paulownia. However, poor removal of hemicellulose (49.92%) by alkali method leads to poor enzymatic hydrolysis [6]. The pretreatment performance was also assessed with regard to changes in the inhibition constant KI during enzymatic hydrolysis. On the whole, there are three types of product inhibition, competitive, uncompetitive and noncompetitive [31]. The inhibition type and its constant can be a result of the substrate material, source of enzyme complex, substrate to enzyme ratio, and hydrolysis time employed. As shown in Table 1, all of three KI values are small and similar and fallen in the range of competitive type (0.05 ~ 6 g/L). This indicates that glucose exerts competitive inhibition to the enzymatic hydrolysis of pretreated Paulownia [31]. The same conclusion is drawn from sugarcane bagasse hydrolysis (KI = 3.34 g/L) after it conducted by steam exploded (and 4% NaOH impregnation) [33]. Moreover, a small KI value of all experiments indicates that glucose can difficultly connect with the zymoprotein. Previous study suggests that, compared to DA pretreatments, AL method has less sugar degradation, and the formation of furan derivative should be avoided [21]. So it is generally believed that a small KI value from DA pretreatment should be attributed to glucose-degradation reactions and the formation of byproducts, such as hydromethylfurfural or furfural. [34]. However, the steric hindrance and probable competition during enzymatic hydrolysis are caused by byproducts deriving from AL pretreatment [35]. Further investigation is needed to confirm inhibitive abilities of the

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byproducts. It’s necessary to reduce the inhibition of glucose product as far as possible to improve the hydrolysis of cellulose. Methods of minimizing the product inhibition include extraction of reducing sugars from hydrolysate during enzymatic hydrolysis and simultaneous saccharification and fermentation, in which the reducing sugars are instantly converted to ethanol or other chemicals [15,36]. UA pretreatment is the best method for enzymatic hydrolysis according to competitive inhibition MichaelisMenten equation, followed by DA and AL pretreatment. As expected, the experiment data and model fits well (Fig. 2). Although UA treatment shows the highest enzymatic hydrolysis efficiency, its industrial application should be carefully evaluated due to its high energy consumption.

4. Conclusion Kinetics of enzymatic hydrolysis of Paulownia by three pretreatment methods was studied under optimal conditions. The competitive inhibition Michaelis-Menten kinetics model was successfully applied to the kinetics study of enzymatic hydrolysis of Paulownia by DA, AL and UA pretreatments. Ultrasonic treatment of Paulownia with the presence of alkali is more efficient than DA and AL pretreatments.

Acknowledgements The research was financially supported by grants from Guangdong Province Science and Technology Project (No: 2013B090200016, 2013B021000008), Joint Fund of Guangdong Province (No: U1401235) and Electric Power Research Institute of Guangdong Grid Co. (No: K-GD20130501).

Nomenclature C t Rmax KM Cs KI Cso dC ------dt

: : : : :

The reducing sugar concentration (g/L) Reaction time (h) Maximum reaction velocity (g/L/h) Michaelis-Menten constant (g/L) Cellulose concentration of enzymatic hydrolysis (g/L) : Inhibition constant (g/L) : Initial cellulose concentration of enzymatic hydrolysis of pretreated Paulownia (g/L)

: Reaction rate (g/(L/h))

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