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Dilute sulphuric acid pretreatment and enzymatic hydrolysis of Jatropha curcas fruit shells

for ethanol production

Pre-print of the paper published in Industrial Crops & Products 53 (2014) 148–153

Published Journal Article is available at http://dx.doi.org/10.1016/j.indcrop.2013.12.029

Ariel Garcíaa,b, Cristóbal Carab, Manuel Moyab, Jorge Rapadoa, Jürgen Pulsc, Eulogio Castrob,

Carlos Martína,c*

aDepartament of Chemistry and Chemical Engineering, University of Matanzas, 44740 Matanzas, Cuba; bDepartament of Chemical, Environmental and Materials Engineering, University of Jaén, 23071 Jaén, Spain;

cTI-Institute for Wood Research, 21031 Hamburg, Germany

*Corresponding author (current address: Department of Chemistry, Umeå University, SE-901 87, Umeå,

Sweden; E-mail: carlos.martin@chem.umu.se; Phone: +46 90 786 54 67)

Abstract

Jatropha curcas is a promising source of oil for the biodiesel industry, and the shells of its fruits

could be considered for ethanol production. In this work, the composition of J. curcas shells is

investigated, and the potential of dilute-sulphuric acid pretreatment for improving the enzymatic

hydrolysis of cellulose is evaluated. A Box-Behnken experimental design was used for assessing the

effect of temperature (110-150°C), H2SO4 concentration (0.5-2.5%) and pretreatment time (15-45

min) on the formation of sugars during pretreatment and on the enzymatic conversion of cellulose.

Cellulose conversions above 80% were achieved both in the separated enzymatic hydrolysis and in

the simultaneous saccharification and fermentation of the pretreated materials. Optimal SSF

conversions were predicted for pretreatments at low temperature (136°C) and moderate acid

concentrations (1.5%) and reaction time (30 min). The inclusion of an extraction step prior to the

pretreatment revealed a further improvement of the enzymatic conversion of cellulose.

Keywords: Jatropha curcas shells, acid pretreatment, enzymatic hydrolysis, lignocellulosic

materials

1. Introduction

The concerns on environmental pollution and on the possible depletion of fossil fuels has

considerably increased the interest for biodiesel and bioethanol as potential substitutes of

petrodiesel and gasoline, respectively (Agarwal, 2007). However, the use of cereals and edible oils

as raw materials could restrict the expansion of the biofuel industry. Therefore, alternative

feedstocks, such as lignocellulosic bioresources and non-edible oils should be utilized for

production of ethanol (Wyman, 2008) and biodiesel (Azam et al., 2005), respectively. The potential of different non-edible oilseeds is being investigated in Cuba (Martín et al., 2010;

Hernández et al., 2013), and Jatropha curcas is one of the most attractive options. The feasibility of

J. curcas as a source of oil has been demonstrated, and there is an increasing interest on its use for

biodiesel production (Koh et al., 2011).

Prior to extraction of the oil, J. curcas seeds should be separated from the fruits, and residual shells

are generated in that operation. The amounts of generated shells are rather high (up to 8 t/ha)

considering that they represent 31.6% of the weight of the fruits (Martín, unpublished), and that J.

curcas can yield up to 25 tons of fruits per hectare (Kumar et al., 2003). Different uses have been

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proposed in order to deal with such a voluminous residue. For example, due to their high calorific

value (between 16-17 MJ/kg), J. curcas shells are attractive for energy applications (Kratzeisen and

Müller, 2009). They can also be used for activated charcoal production (Ramakrishnan and

Namasivayam, 2009). The possibility of producing ethanol from J. curcas shells and other co-

products resulting from biodiesel production was recently reported (Visser et al., 2011). The in situ

produced ethanol could be directed, together with the oil, to biodiesel production in an integrated

configuration. As shown by Gutiérrez et al. (2009), the integration of ethanol and biodiesel

production processes using a single source of biomass would allow considerable reduction of the

energy costs compared with the autonomous production of each of them, and would lead to

decrease of the generation of solid wastes.

In order to obtain sugars that could be fermented to ethanol, the polysaccharides contained in

lignocellulosic materials, such as J. curcas shells, should be hydrolysed by means of acids or

enzymes (Taherzadeh and Karimi, 2007). Enzymatic hydrolysis produces higher sugar yields and

lower decomposition products than acid hydrolysis, but it requires a pretreatment step in order to

make cellulose macromolecules accessible to cellulolytic enzymes. Different pretreatment methods

have been investigated, and the leading technologies are discussed and described elsewhere (Yang

and Wyman, 2008). Dilute-acid prehydrolysis is a pretreatment method that has resulted effective

for different materials, and that has high potential for industrial application (Mosier et al., 2005;

Rocha et al., 2011). Due to that this method leads to high recovery (80-90%) of the hemicellulosic

sugars upon pretreatment, and to high cellulose conversion (above 90%) upon enzymatic

hydrolysis, it is favoured by the National Renewable Energy Laboratory (NREL), which is leading

the largest cellulosic ethanol development effort in the world (Yang and Wyman, 2008).

The reports on pretreatment and hydrolysis of J. curcas shells are rather scarce in the literature. In

the first reported work, the pretreatment of J. curcas shells at 121°C for 1 h with either 0.5% H2SO4

or 1% NaOH led to rather low glucan conversion (40-41%) (Visser et al., 2012). In another recent

publication, Marasabessy et al. (2012) reported the pretreatment of J. curcas shells in the range of

140-180°C with 0.1-0.9% H2SO4, and showed that the highest glucan conversion upon

simultaneous saccharification and fermentation (SSF) can be achieved for the material pretreated at

178°C, 0.9% H2SO4 and 30 min. In the current work, we used slightly higher acid concentrations

than those used by Marasabessy et al. (2012) in order to assess the possibility of obtaining

comparable hydrolytic conversions at lower temperature. Additionally, we investigated not only

simultaneous saccharification and fermentation, but also the separate hydrolysis scheme.

2. Materials and methods

2.1. Materials

J. curcas fruits, harvested in June, 2011 in a two-years plantation (Granja Paraguay, Guantánamo,

Cuba), were collected manually, air dried to 85-90% dry matter content and peeled in a knife-mill

(Retsch, SM 100, Haan, Germany). The shells were dried at 50°C for 24 h, milled to 2-mm particle

size, sieved to remove the rejects, and kept in plastic bags at room temperature until further use. A

small fraction of the shells was screened through a 1-mm sieve and used for the raw material

analysis.

2.2. Pre-extraction of the raw material

Milled J. curcas shells corresponding to 70 g dry matter (DM) were suspended in 700 mL of water

in a 1-L blue-cap bottle. The bottle with the suspension was heated at 120°C for 1 h in an autoclave

(EZE-Seal, Parker Autoclave Engineers, Erie, PA). After that, the solid material was separated

from the extract by vacuum filtration, and the filter cake was washed with water, and dried for 5

days at room temperature until a final dry matter content of approximately 94%.

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2.3. Pretreatment

The pretreatment was performed in a 1-L reactor (Parr Instruments Company, Moline, IL) heated

with an external electrical mantle. A Box-Behnken response surface experimental design was

applied to evaluate the effect of the temperature (110-150°C), the sulphuric acid concentration (0.5-

2.5%) and the pretreatment time (15-45 min) on the enzymatic hydrolysis. The selection of the

factors and their ranges were based on preliminary results by this group (García et al., 2012). J.

curcas shells (50 g dry matter) were suspended in a dilute-sulphuric acid solution giving a 500 g

reaction mixture. The reaction batch was heated to the work temperature (heating time, 10–15 min),

and held for the time indicated in the experimental design. The temperature was monitored by an

internal thermocouple, and controlled with a PID module. After elapsing the reaction time, the

reactor was cooled to approximately 30-35°C (cooling time, 15–20 min) by immersion in an ice

water bath. In an additional experiment, triplicate pretreatments of raw and pre-extracted J. curcas

shells were carried out at 165°C with 1% H2SO4 and for 35 min.

2.4. Enzymatic hydrolysis

An aliquot (1.25 g) of the dried pretreated solids was suspended in 25 mL of 0.1 M citrate buffer

(pH 4.8). A commercial preparation of Trichoderma reesei cellulases (Celluclast 1.5L) and a -

glycosidase preparation (Novozym 188), both kindly donated by Novozymes A/S (Bagsværd,

Denmark), were used. The cellulases were added at a loading of 15 FPU/g DM, and the -

glycosidase as 15 IU/mL. The reaction mixture was incubated in an orbital shaker (Certomat-IS,

Braun, Germany) at 50°C and 150 rpm for 48 h. At the end of the hydrolysis, glucose was

quantified by HPLC, and the results were used for calculating the enzymatic convertibility of

cellulose. All of the experiments were performed in duplicates.

2.5. Simultaneous saccharification and fermentation

Suspensions of 1.25 g of the pretreated solids in 24 mL of citrate buffer (pH 4.8) were prepared in

Erlenmeyer flasks. The suspensions were supplemented with yeast extract (5 g/L), NH4Cl (2 g/L),

KH2PO4 (1 g/L) and MgSO4·7H2O (0.3 g/L). Enzymes were added in the same amounts as for the

enzymatic hydrolysis. A Saccharomyces cerevisiae culture (1 mL) was inoculated, and the reaction

mixture was incubated at 35°C and 150 rpm for 72 h. All of the experiments were performed in

duplicates.

2.6. Analytical methods

The content of moisture, mineral components, extractive compounds, structural carbohydrates and

lignin in the raw J. curcas shells and in the pretreated materials was analysed according to NREL

analytical procedures. Moisture was determined gravimetrically after drying the material at 105°C

(Sluiter et al., 1998a). Mineral components were determined as ash after incineration of an aliquot

of the material at 550°C (Sluiter et al., 1998b). Extractives were determined by consecutive

extractions with water and ethanol (Sluiter et al., 1998d). The extractive-free material obtained after

water and ethanol extraction was submitted to analytical acid hydrolysis for determination of the

structural carbohydrates and lignin. The released sugars were quantified chromatographically, and

lignin was determined gravimetrically (Sluiter et al., 1998c). The quantification of the sugars in the

analytical acid hydrolysates was performed by HPLC (Varian Scientific Instruments, Palo Alto,

CA). Glucose, xylose, arabinose, galactose and mannose were separated with an Aminex HPX-87H

column (Bio-Rad, Hercules, CA) at 65°C using sulphuric acid (4 mM) as mobile phase at a flow

rate of 0.6 mL/min, and were detected with an RI detector. The content of total reducing sugars in

the prehydrolysates obtained in the pretreatment was determined using the dinitrosalicylic acid

method (Miller, 1959). The experimental results were statistically processed using the softwares

Statgraphics Plus 5.0 and Design-Expert ® 7.0.

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3. Results and discussion

3.1. Chemical characterisation

The results of the chemical characterization are shown in Table 1. The content of glucans (32.8%) is

comparable to that of other lignocellulosic materials (Martín et al., 2006), and it is higher than the

content reported for J. curcas shells from Indonesia (20.4%) (Marasabessy et al., 2012) and from

Brazil (26.5%) (Visser et al., 2012). An interesting feature of the carbohydrates contained in J.

curcas shells is the relatively high overall content of hexosans (37.7% of the dry matter content

considering the sum of glucans, mannans and galactans). Because of that, J. curcas shells could be

considered a potential feedstock for ethanol production. The hydrolysis of these hexosans render

glucose, mannose and galactose, which are fermentable by S. cerevisiae, the main ethanologenic

organism used in the industry (Martín and Jönsson, 2003).

Component Content, % (w/w)

Glucans 32.8 (2.2)

Xylans 8.3 (0.3)

Arabinans 0.9 (0.0)

Mannans 2.7 (0.0)

Galactans 2.2 (0.0)

Lignin 19.6 (0.2)

Water extractives (WE) 32.7 (1.3)

Sugars in WE* 5.8 (0.2)

Ethanol extractives 4.0 (0.3)

Minerals 14.4 (1.4) Table 1. Chemical composition of J. curcas shells

*Determined as total reducing sugars

Lignin content is comparable to that reported for shells of Brazilian J. curcas (Visser et al., 2012),

and it is within the typical range observed for other biomass materials (Martín et al., 2006).

However, it is considerably higher than the one reported for Indonesian samples (Marasabessy et

al., 2012). On the other hand, the ash content was rather high, and compares only with a few

materials, such as rice hulls (López et al., 2010) and barley husks (Garrote et al., 2004). It should be

remarked that ash determination was performed in parallel, but not sequentially, with the analyses

of extractives, carbohydrates and lignin. Part of the components accounted as ash might have been

solubilised during the water extraction and the analytical acid hydrolysis. Therefore, it is not correct

to include the ash content in a summative analysis together with the other biomass components.

It is noteworthy the high amount of water extractives (32.7%) in the raw material. The sugars

contained in the fraction of water extractives reached 5.8% of the dry weight of the raw shells.

Although they could also be of interest for ethanol production, no prediction of their fermentability

by S. cerevisiae can be made since their analysis in this work was restricted to the quantification of

total reducing sugars. A further chromatographic identification of the individual sugars is required.

Another interesting observation was the color of the water extract. It was dark brown at pH 4 - 7,

and it turned light brown when the pH was adjusted to 1 - 2. It would be of interest to identify the

colored components contained in that fraction.

3.2. Dilute-acid pretreatment

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As can be seen from the yields of pretreated solids, which ranged from 51.1 to 75.3% (Table 2), the

dilute-acid pretreatment led to the solubilisation of between one quarter and one half of the raw

material. The analysis of significance of the independent factors showed that the yield of solids

significantly decreased with the temperature, the sulphuric acid concentration and their interaction,

whereas it significantly increased with the quadratic effect of the pretreatment time (Fig. 1-A).

Indeed, the yield of pretreated solids was above 70% at 110ºC, but it decreased to 51.1-65.5% in the

pretreatments performed at 150°C. The decrease of the yield with the increase of sulphuric acid

concentration is evident when comparing the pairs of experimental runs performed with different

concentrations but at the same temperature and pretreatment time. For example, for pretreatments 1

and 4, carried out at 110ºC and 30 min, the yield in the experimental run 4 (70.7%), in which the

H2SO4 concentration was 2.5%, was lower than in the number 1 (73.4%), which was performed

with 0.5% H2SO4. The same was true for the experiments 5 and 10 (130°C for 15 min), 6 and 11

(130°C for 45 min), and 12 and 15 (150°C for 30 min). The yields of solids in all the experiments at

110°C and in those at 130°C performed with the lowest acid concentration are comparable with the

result (72.9%) reported previously for pretreatment of J. curcas shells at 121°C with 0.5% H2SO4

(Visser et al., 2012).

+-

0 4 8 12 16 20 24

AA

AB

B:Time

BB

AC

C:acid conc

A:Temperature

Figure 1. Yield of pretreated solids after pretreatment. A, Analysis of significance of the independent factors; B,

Estimated response surface for the effect of temperature and sulphuric acid concentration.

The effect of temperature and sulphuric acid concentration on the yield of solids is very well

illustrated in the diagram of response surface (Fig. 1-B). Although, in general, the yield of solids

also decreased when the pretreatment time rose from 15 to 45 min, that trend was noticed mainly in

the pretreatments performed at 130 and 150ºC, but not in those carried out at 110ºC (Table 2), and

Temperature

H2SO4 conc110 120 130 140 150

00.5

11.5

22.5

45

55

65

75

85

A

B

6

the effect was not significant (Fig. 1-A). This can clearly be shown when comparing the

experimental runs 5 and 6 (130°C, 0.5% H2SO4), 10 and 11 (130°C, 2.5% H2SO4), and 13 and 14

(150°C, 1.5% H2SO4). On the other hand, in the runs 2 and 3, performed under identical

temperature (110°C) and H2SO4 concentration (1.5%) but for different pretreatment time, the same

yield was obtained. Based on the experimental results, the following model (Eq. 1, R2 = 94.3%),

showing the effect of the temperature (T), pretreatment time (t), sulphuric acid concentration (conc)

and their interactions on the yield of solids, was developed: 2t5.8 conc T5.6 conc 9.1T 14.164.4solids of Yield (Equation 1)

No Temperature, °C H2SO4 concentration,

% (w/w)

Time, min Yield of

solids, %

1 110 0.5 30 73.4

2 110 1.5 15 75.3

3 110 1.5 45 75.3

4 110 2.5 30 70.7

5 130 0.5 15 73.3

6 130 0.5 45 69.6

7 130 1.5 30 65.0

8 130 1.5 30 64.0

9 130 1.5 30 63.4

10 130 2.5 15 64.2

11 130 2.5 45 59.0

12 150 0.5 30 65.5

13 150 1.5 15 62.4

14 150 1.5 45 58.8

15 150 2.5 30 51.1

Table 2. Box-Behnken experimental design used for investigating the acid pretreatment of J. curcas shells, and yield of

solids obtained.

The negative signs of the coefficients of temperature and acid concentration reflect the decrease of

the recovery of pretreated solids with the increase of those variables. On the other hand, the sign of

the interaction means that using high temperature requires lower acid concentrations and vice versa

in order to avoid the decrease of the yield. As the quadratic time is significant, the response surface

diagram features some curvature (Fig. 1-B). The model predicted an optimal yield of 77.5% for the

pretreatment performed at 110.2°C, 0.5% H2SO4 and for 15.1 min.

Glucan and lignin content in the pretreated solids increased with the raise of temperature, H2SO4

concentration and pretreatment time (Fig. 2-A). Xylan content dropped with the increase of all the

experimental variables as a result of its partial hydrolysis. Mannans and galactans were also mostly

solubilised.

As a result of the partial solubilisation and hydrolysis of xylans, mannans and arabinans, simple

sugars were formed. As shown in the response surface diagram (Fig. 2-B), the concentration of total

reducing sugars in the pretreatment liquors increased with the increase of the temperature and the

sulphuric acid concentration. For example, for the experiments performed at 150°C and 30 min, the

increase of the concentration of sulphuric acid from 0.5 to 2.5% led to a sharp increase of the sugar

concentration from 7 to 20.6 g/L. These results are in good agreement with the values achieved by

Marasabessy et al. (2012), who reported increasing release of pentoses and hexoses with increasing

of the temperature and the sulphuric acid concentration.

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A model based on the experimental results (Eq. 2, R2 = 93.3%) predicted an optimal concentration

of reducing sugars of 23.4 g/L for the pretreatment performed at 149.4°C and 2.4% H2SO4 for 45

min. 2T27.4 conc 9.7T 10.915.0sugars reducing Total (Equation 2)

The pretreatment time was not included in the model since the analysis of significance revealed that

it did not exert any significant effect. This is consistent with previous findings about the effect of

temperature and acid concentration on degradation of pentoses to furfural during acid pretreatment

of J. curcas shells (Marasabessy et al., 2012).

T ime=30.0

TemperatureH2SO4 conc

110 120 130 140 1500

0.51

1.52

2.5

-10

0

10

20

30

Figure 2. Analysis of the pretreated material. A, content of the main components in the solid fraction (%); B, estimated

response surface for the effect of temperature and sulphuric acid on the concentration of total reducing sugars (g/L) in

the liquid fraction.

3.3. Enzymatic hydrolysis

The enzymatic conversion of cellulose in the pretreated solids was investigated by two approaches,

namely separated hydrolysis (SH) and simultaneous saccharification and fermentation (SSF). The

analysis of variance showed that the responses of both hydrolysis schemes were strongly affected (P

< 0.01) by the temperature and the quadratic effect of acid concentration. Additionally, the

enzymatic conversion of cellulose in the separate hydrolysis was affected by the sulphuric acid

concentration, while the SSF conversion was influenced as well by the acid concentration and

quadratic effects of time and temperature. Models predicting the effect of the pretreatment

conditions on the enzymatic conversion in both hydrolysis schemes were proposed (Eq. 3 and 4). 2conc8.0 conc14 T 9.1- 85.3 conversion SH (Equation 3)

A

B

8

2t18.3 2conc8.3 -conc5.0 2T11.3 T8.0 81.3 conversion SSF (Equation 4)

The cellulose-to-glucose and cellulose-to-ethanol conversions achieved in the SH (Fig. 3-A) and

SSF (Fig. 3-B), respectively, were high, with several individual experimental runs reaching values

above 80%. In general, the best conversions were achieved for the pretreatments performed at

intermediate severity, whereas the materials pretreated at either low or high values of the

independent factors gave rise to values below 70% of the theoretical yield. In the separate

hydrolysis, the developed model (Eq. 3) predicted an optimal cellulose-to-glucose conversion

(87.1%) for the material pretreated at 134°C, 1.0% H2SO4, and for 32.4 min. Meanwhile, the model

developed for the SSF experiments (Eq. 4) predicted a maximal cellulose-to-ethanol conversion

(82.0%) for the material pretreated at 136°C, 1.5% H2SO4 and for 30 min. Since the SSF

conversions achieved in this work were higher than the optimal glucan yield reported for the

pretreatment performed at 178°C, 0.9% H2SO4, and 30 min (Marasabessy et al., 2012), one can

conclude that low temperature pretreatments (around 135°C) combined with moderate acid

concentrations (1.5%) are more favourable for activating the cellulose contained in J. curcas shells

than high temperature pretreatments (around 180°C) at lower acid concentration (1%). This result

has economic implications due to the energy saved in heating the reactor.

TemperatureH2SO4 conc

110 120 130 140 1500

0.51

1.52

2.5

55

65

75

85

95

Temperature H2SO4 conc

110 120 130 140 1500

0.51

1.52

2.5

55

65

75

85

Figure 3. Estimated response surface for the effect of temperature and sulphuric acid concentration on the cellulose-to-

glucose conversion of the separated hydrolysis (A) and on the cellulose-to-ethanol conversion in the SSF (B).

A

A

B

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3.4. Pretreatment with preliminary water extraction

The high content of extractives revealed by the raw material analysis can affect the processing of

the J. curcas shells. This fact has been reported in other lignocellulosic materials. For example,

Ballesteros et al. (2011) stated that enzymatic hydrolysis of pretreated olive tree pruning could be

affected by condensation reactions between extractives and acid insoluble lignin during

pretreatment. The removal of water-soluble extractives prior to pretreatment could improve the

pretreatment effectiveness and save the carbohydrates contained in that fraction, which otherwise

would be destroyed during high temperature processing of the raw material. In order to confirm the

above-stated hypothesis, an additional experiment was performed. The water extracts were removed

by an extraction step at 120°C, and the resulted solids were submitted to pretreatment in parallel

with a sample that was not subjected to preliminary extraction. The experiment was performed at

165°C, with the aim of comparing the effect of the pre-extraction at a temperature between our

optimum and the optimum reported by Marasabessy et al. (2012).

Figure 4. Results of the pretreatment with (white columns) and without (black columns) preliminary removal of water

extractives.

The solids resulting from the pretreatment of the pre-extracted J. curcas shells contained less lignin

and displayed better enzymatic conversion than those resulting from the direct pretreatment of the

raw material (Fig. 4). The higher Klason lignin content of the non-extracted solids might be due to

the formation of acid-insoluble by-products as result of the degradation of sugars and some other

components of the extracts during pretreatment. The better enzymatic conversion of the pre-

extracted material might be related with the removal of potential cellulase inhibitors contained in

the water extracts. The preliminary extraction of the raw material also led to a higher concentration

of reducing sugars in the pretreatment liquors.

4. Conclusions

The high cellulose-to-ethanol conversion achieved by both separate enzymatic hydrolysis and

simultaneous saccharification and fermentation of acid-pretreated J. curcas shells shows the

feasibility of low temperature (around 135°C) pretreatment combined with moderate acid

concentrations (1.5%) for activating the cellulose contained in J. curcas, and compares favourably

with previously reported results at higher temperature 180°C and lower acid concentration. The

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potential of a preliminary extraction step for improving the effectiveness of the dilute acid

pretreatment of J. curcas shells was also demonstrated.

Acknowledgments

The experimental work was financially supported by Estancias de Excelencia, Junta de Andalucía

(Spain). The financial support given by the Alexander von Humboldt Foundation and by the Swiss

Agency for Development and Cooperation through the BIOMAS-Cuba project is also gratefully

acknowledged.

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