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Steam explosion as pre-treatment of Cynara cardunculus prior to delignification Ana Lourenço, Jorge Gominho, Maria Dolores Curt, Esteban Revilla, Juan Carlos Villar, and Helena Pereira Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.6b03854 • Publication Date (Web): 09 Dec 2016 Downloaded from http://pubs.acs.org on December 13, 2016

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Industrial & Engineering Chemistry Research

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Steam explosion as pre-treatment of Cynara cardunculus prior to delignification

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Ana Lourençoa*, Jorge Gominhoa, Maria Dolores Curtb, Esteban Revillac, Juan Carlos Villarc, and Helena Pereiraa

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a

Centro de Estudos Florestais, Instituto Superior de Agronomia, Universidade de Lisboa, Tapada da Ajuda, 1349-017 Lisboa, Portugal.

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b

Escuela Técnica Superior de Ingenieros Agrónomos, Universidad Politécnica de Madrid, Av. Complutense s/n, 28040 Madrid, Spain.

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c

Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Centro de Investigación Forestal, Carretera de la Coruña, km 7.5, 28040 Madrid, Spain.

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*Corresponding author: [email protected]

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Abstract

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Cardoon (Cynara cardunculus) stalks were submitted to steam explosion (183ºC, 5 min) with a yield of 88%, and a lignin loss of around 7%. Cardoon (C) and steam exploded cardoon (CSE) were characterized by wet chemical analysis and Py-GC/MS. Total lignin and its S/G ratio were 21.6% and 1.35 in C; and 22.4% and 1.40 in CSE. These samples were delignified by kraft and organosolv processes. Cardoon pulps were obtained with different yields of 45.1% for kraft and 61.2% for organosolv. Kraft pulps yields were 45.1% and 50.9% respectively for Ck and CSE-K, corresponding to a lignin loss of 74.7% and 72.2%. The S/G ratio was similar 1.25 and 1.23. Organosolv pulps presented higher yields (61.2% vs. 73.3%), were less delignified (30.5% vs. 13.8%), and the S/G ratio was 1.54 and 1.47 respectively for Corg and CSE-org. Overall, the condition chosen for steam explosion allowed a low mass loss, but did not promote delignification and neither enhanced lignin removal during kraft and organosolv processes.

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Keywords: Cardoon, pre-treatment, organosolv, kraft, Py-GC/MS, S/G ratio

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Introduction

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The fragmentation of biomass has seen an enormous research increase in recent years in the context of a potential application in biorefineries. Most studies aimed at sugars recovery, having in mind the production of bioethanol or cellulose1 but lignin is also a research target since its removal is essential for the efficient enzymatic or chemical hydrolysis of polysaccharides. Lignin by itself is equally important for biomass valorization since it plays an important role in the production of biomaterials and chemicals e.g. polymers and resins2.

48 49 50 51 52

Different pre-treatments have been tested for biomass fractionation. Steam explosion is such a pre-treatment, by which biomass changes from being a tenacious flexible material into a brittle rigid material3. Steam explosion promotes the breakdown of the biomass matrix, making the structural polymers more accessible for subsequent treatments such as hydrolysis or fermentation1,4.

53 54 55 56 57 58 59 60 61

Two types of reactions occur during steam explosion: in the beginning there is a fast depolymerization of lignin and hemicelluloses by acid hydrolysis but as temperature increases, condensation and repolymerization reactions dominate, promoting an increase of acid insoluble lignin5. The hemicelluloses are released from the cell wall matrix, also liberating acetic acid that will enhance hydrolysis and further sugar reactions; for example, xylose is mainly degraded into furfural6. Cellulose molecules are deconstructed and degraded to some extent into hydroxymethylfurfural while lignin is released from the cell wall mainly by cleavage of β-O-4 linkages7. Condensation reactions can also occur, and fragments can be deposited onto the fibers surface4,8.

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The most important variables during steam explosion are time and temperature as well as chip size since in larger chips the heat transfer is less efficient and they are less 2 ACS Paragon Plus Environment

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degraded in the interior and overcooked in the exterior9. Therefore, chip size is usually small e.g. 8 to 12 mm when using softwood biomass; however, since this requires significant amount of energy, the influence of steam explosion on different chip sizes has been evaluated5,9.

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Overall, steam explosion is an attractive biomass pre-treatment since i) it uses only a limited number of chemicals, ii) it requires relatively low energy, and iii) depending on the conditions applied, most of the original cellulose and hemicelluloses-derived carbohydrates can be recovered for fermentation10,11.

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The effect of steam explosion has been reported for several lignocellulosic materials such as wheat straw10, eucalyptus wood4,12 or pine wood5. The objective was mainly to recover sugars for bioethanol production, but also to increase the heating value, bonding properties and hydrophobicity before pelletization13.

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Cynara cardunculus (cardoon) has also been pre-treated by steam explosion and the results showed an improvement of the raw-material for different purposes: i) increased the accessibility of residual polysaccharides towards enzymatic hydrolysis for bioethanol production14,15; ii) cellulose nanocrystals obtained from steam treated cardoon could reinforce filler in paper sheets with better mechanical strength16; iii) the binderless fibreboards produced from steam exploded cardoon presented improvements on the physical properties of the boards17.

83 84 85 86 87 88

Cardoon is an interesting biomass since it has a high production potential and is a multifunctional crop with different applications18,19,20. The cellular features and chemical composition of the cardoon stalks have been investigated and they were found to have an interesting pulping potential by delignification21,22,23. Lignin content in cardoon is around 19.0% and has a composition of mainly syringyl and guaiacyl lignin units24,25.

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The effect of steam explosion upon the cardoon lignin has not been explored nor on its effects on subsequent delignification. This is the objective of this study where an evaluation of steam explosion as a pre-treatment of cardoon biomass previous to delignification is made using two pulping processes (kraft and organosolv). The initial and the residual lignin in the pulped samples were analysed using analytical pyrolysis.

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Experimental

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Sampling

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The plant material came from an experimental crop of Cynara cardunculus L. var. altilis established from micro-propagated plants at the Technical University of Madrid, Spain (40º24’40”N, 03º40’41”W, 667 m a.s.l.). After plant establishment, the crop was maintained under rainfed conditions, following a perennial cultivation system with annual harvests of the aboveground biomass. The climate is continental-Mediterranean, with 438 mm annual rainfall, with 13.9ºC of mean temperature, reaching 39.1ºC of absolute maximum temperature in July, and -10.1ºC absolute minimum temperature in January, and a 4-month dry period in summertime.

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The stalk fraction including stems and branches of the 4th year crop harvest was collected for this study, air dried until a moisture content of 7% of dry matter, and milled in a knife mill (Black Decker gs-1800, UK) for size reduction. The triturated material contained particles of different sizes including up to ca. 2 cm long fibrous bundles, medium sized 0.5 to 1 cm particles and fine particles below 0.5 mm (Figure 1.left).

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Steam explosion

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The steam explosion pre-treatment of cardoon was conducted in a 26 L stainless steel digestor (Cadepla S.L., Valdemoro, Spain), connected to a blowing tank, where the material was discharged after the treatment. A charge of 2.05 kg of cardoon was steamed in the digestor during 5 min at 183 ºC, at maximum pressure of 7.8 MPa, and discharged at 5.9 MPa into the blowing tank. The solid residue named as CSE (cardoon after steam explosion) was washed with cold water, air-dried, and weighed. Two samples were taken and dried at 105 ºC until constant weight to determine the content of water and calculate the total solid yield after the steam explosion.

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The severity factor of the steam explosion pre-treatment was calculated according to equation 1

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= . ×

Equation 1

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where T is the temperature (ºC) and t is the duration of the pre-treatment (min)26. The severity factor of the steam explosion treatment was 3.14.

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Colour measurements

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The colour of a milled sample (40-60 mesh) of cardoon and of the steam exploded cardoon were characterized by the CIE L*a*b* scale with a Minolta CM-3630 spectrophotometer. Three measurements were made and the mean value calculated. The colour differences between CSE and C were calculated as ∆L*, ∆a*, ∆b* and the ∆E was calculated as equation 2

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∗

∗

∗

ΔE = − ∗ ) + ! − !∗ ) + " − "∗ )

Equation 2

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Delignification experiments

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The untreated cardoon chips (C) and the steam-exploded cardoon chips (CSE) were used in two delignification experiments using a 11:1 liquor-to-solid ratio: kraft and organosolv (ethanol:water). The conditions used were: i) for the kraft process (CK and CSE-K) 18% active alkali and 25% sulfidity; ii) for the organosolv process (COrg and CSEOrg) a solution of 33% ethanol in water (v/v) with 0.1% sulfuric acid (v/v).

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The delignification experiments were conducted in four reactors (each with 1 L). The reactors were placed in a 20 L rotatory pressurized vessel, containing hot water to heat the reactors and indirectly heat the cooking liquor. The temperature of the vessel was maintained at 160 ºC, controlled by computer software. The time needed to reach the maximum temperature was 40 min and the maximum temperature was maintained for 45 min to obtain an H factor of 330.

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The reactors were removed from the vessel, and the delignified material was washed with distillated water, defiberized in a laboratory pulper MK.IIIC (Messmer Instruments Kent, United Kingdom), dried at room temperature and total yield determined. Two batches were made for each delignification process and sample type, and the average yields were calculated.

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Chemical characterization

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The solid samples of initial cardoon (C), steam-exploded cardoon (CSE) and the delignified samples (CK, COrg, CSE-K and CSE-Org) were milled in a knife mill (Retsch SM 2000, Frankfurt, Germany), passing through a sieve of 1 mm x 1 mm, and separated in a vibratory sieve (Retsch AS 200, Frankfurt, Germany) to collect the 40-60 mesh fraction for chemical analysis.

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Chemical characterization was made using adaptations to TAPPI standards: ash content (T15 os-58), extractives content by successive extraction with dichloromethane, ethanol and water (T264 om-88), Klason and soluble lignin (T222 om-88 and UM250 om-83, respectively). Neutral monosaccharide composition was determined in the hydrolysate from the lignin analysis (T249 cm-00). The monossacharides were separated by a Dionex ICS-3000 by HPLC, using an Aminotrap plus Carbopac SA10 column, and the results reported as percentage of total monosaccharides. Holocellulose was determined according to a modified chlorite method27. The samples CSE, CK, Corg, CSE-K and CSE-Org were extracted with water during 48 h for the removal of soluble compounds prior to chemical and pyrolysis analysis. All determinations were made in duplicate samples.

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Gas chromatography-mass spectrometry (GC-MS)

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The lipophilic extractives obtained by solubilization in dichloromethane of cardoon and cardoon after steam explosion were analyzed by GC-MS. For the analysis, 1 mg of the extract was weighed, suspended in 120 µL of pyridine (Sigma–Aldrich) and derivatized with 80 µL of BSTFA (N,O-bis(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane (Sigma–Aldrich)). This mixture was kept at 60 ºC during 30 min. Afterwards, 3 µL of the TMS extract was injected in a GC-MS Agilent (7890A-5975C 5 ACS Paragon Plus Environment


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MSD) in splitless mode. The GC had a high temperature capillary column (with a polyamide-coated fused silica phase): Zebron 5HT (30m x 0.25 mm x 0.1 µm film thickness). Helium was used as carrier gas at a constant flow of 1 mL min-1. The oven temperature was programmed as follows: 100 ºC (1 min); increased to 150 ºC at a rate of 10 ºC min-1; to 200 ºC at a rate of 5 ºC min-1; to 300 ºC at a rate of 4 ºC min-1; and finally to 380 ºC (5 min) at 10 ºC min-1. The injector temperature was maintained at 280 ºC and the interface at 330 ºC. Compounds were identified by comparing their mass spectra with Wiley, NIST libraries, by mass fragmentation and in some cases with authentic standards. If two compounds were overlapped, each area was allocated proportionally by the area of each base peak. The relative abundance of each was calculated using its peak area in the total ion current (TIC) chromatogram, and expressed as milligrams per gram of dry weight extract, as average of two replicates.

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Analytical pyrolysis (Py-GC/FID)

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The samples were characterized by analytical pyrolysis after being washed with hot water. The samples were dried and powdered in a Retsch MM200 mixer ball mill during 10 min, and 0.20 mg of each sample was pyrolysed in a 5150 CDS apparatus linked to an Agilent GC 7890B with a mass detector system 5977B installed with a ZB-1701 fused-silica capillary column (60 m x 0.25 mm i.d. x 0.25 µm film thickness). The chromatograph oven program was: 40 ºC, held for 4 min, 10 ºC min-1 to 70 ºC, 5 ºC min-1 to 100 ºC, 3 ºC min-1 to 265 ºC, held for 3 min, 5 ºC min-1 to 270 ºC, held for 9 min. The compounds were identified comparing their mass spectra with Wiley, NIST2014 and by mass fragmentation. The peak molar area of each compound identified was calculated, summed and the percentage of each compound calculated. The percentage of guaiacyl (G) and syringyl (S) derived products were separately summed and the S/G ratio calculated.

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X-ray scattering

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A sample of cardoon and of cardoon after steam explosion were milled to powder in a Retsch MM200 mixer ball mill, and dried under vacuum oven over night. For comparison, a sample of microcrystalline cellulose for column chromatography (Macherey-Nagel ref. 81529, Germany) was also tested. A portion of the sample was placed into a sample holder and was hand pressed with a solid plug to create an even horizontal surface and introduced in a X-ray diffractometer (Rigaku Miniflex II). The diffractograms were collected applying a wide angle X-ray diffraction with Cukα radiation generated at 30 kV and 15 mA, and the intensities were measured in the range of 0⁰ < 2ϴ < 60⁰, with a scan speed of 2 deg min-1, 0.02⁰ resolution and run total time of 30 min. The diffractograms were corrected to baseline and the results used for calculation of the crystalline index (CI) following Segal et al.28, as in equation 3

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$% =

' ( )'*+ ' (

× 100

Equation 3


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where I002 is the maximum intensity above the baseline (at approximately 22⁰) and Iam the minimum peak intensity above the baseline (at approximately 18⁰).

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Results

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Chemical characterization of cardoon

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The chemical analysis of cardoon and of pretreated cardoon with steam explosion is presented in Table 1. Cardoon has 7.4 % ash content, and 14.0% total extractives constituted mainly by water and ethanol extracts. On an extractive-free base, total lignin represented 21.6% of the material, holocellulose 86.4% and α-cellulose 55.0%. Polysaccharides include mainly glucose and xylose, with respectively 62.9% and 24.6% of the total monosaccharides, while acetic acid represented 7.0% and the other monosaccharides less than 3.0%. The diffractogram of cardoon is given in Figure 2 and the cellulose crystallinity index was calculated as 43.8%.

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Steam explosion

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The steam explosion pretreatment had a total yield of 88.0%. In comparison to the untreated cardoon, the steam exploded cardoon was characterized by a lower extractives content of 12.4% (Table 1), but with a similar distribution by solvents. Total lignin content was similar but holocellulose decreased to 68.4% while α-cellulose increased to 52.2%. The monosaccharide composition showed mainly an increase of glucose proportion (68% of total) and a small reduction on the proportion of the other sugars as well as of acetic acid.

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The crystallinity index of cellulose in the steam exploded cardoon (Figure 2) increased to 47.9%.

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The colour of cardoon changed with the steam explosion (Figure 1). It became darker with a lightness decrease from 74.2 to 50.9, and an overall colour change ∆E of 23.8.

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GC-MS analysis of lipophilic extracts

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A detailed analysis of the lipophilic extracts of C and CSE was performed by GC-MS. The total ion chromatogram (TIC) for both samples is presented in Figure 3, where the compounds identified are listed by families with the respective amounts (mg g-1 of oven dry extract), while the peak assignment is given in Supporting information S1. The distribution of the chemical families is presented in Figure 4. The percentage of lipophilic compounds identified was in average 75% and 82% of total area, respectively for C and CSE.

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The main families identified in cardoon (S1 and Figure 4) were pentacyclic triterpenes (38%), fatty acids (28%), steryl glycosides (14%) and sterols (10%); alkanes, aromatics, fatty alcohols, saturated di-acids, hydroxyl fatty acids and monoglycerydes were also present in small amounts (Table 2). The main compounds in cardoon lipophilic extract 7 ACS Paragon Plus Environment


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were: hexadecanoic acid (109 mg g-1), β-amyrin (75 mg g-1), taraxasterol (72 mg g-1), sitosteryl-3β-D-glucopyranoside (64 mg g-1), and stigmasterol (33 mg g-1).

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In the steam exploded sample the composition was slightly different: fatty acids were predominant (48%), followed by pentacyclic triterpenes (28%), and sterols (12%) (Figure 4). Therefore, the main compounds in the steam exploded sample were the hexadecanoic acid (109 mg g-1), (9Z,12Z)-octadeca-9,12-dienoic acid (97.8 mg g-1), (Z)octadec-9-enoic acid (83.8 mg g-1), octadecanoic acid (52 mg g-1), taraxasterol (58.7 mg g-1), β-amyrin (47.9 mg g-1), stigmasterol (45.1 mg g-1) and β-sitosterol (43.8 mg g-1).

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Delignification

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The pulp yields, calculated based on the total solids remaining after delignification (C and CSE) are presented in Table 2.

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The kraft pulped cardoon samples produced samples with fibres well dissociated from each other and a pulp visual aspect. The total yield was 45.1%, and a residual lignin of 10.4% thus corresponding to a delignification degree of 75% of the original lignin in cardoon. The organosolv process showed a different performance: the aspect of the delignified samples was similar to that of the starting material with the presence of still undissociated clusters; the yield was higher (61.2%) as well as the residual lignin (21.1%). The organosolv process achieved a delignification of 31%.

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The effect of the steam explosion treatment on delignification was also studied in relation to the two pulping processes. The kraft pulped CSE samples showed a higher total yield of 50.9% but a similar residual lignin of 10.7% that corresponded to a delignification of 72% of the lignin in the steam exploded sample. The organosolv process yielded 73.3% of solids with a high content in lignin (23.0%) and corresponding to a solubilization of 14% of the original lignin.

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The monosaccharide composition was also evaluated and the results are presented in Table 3. Glucose proportion increased with both delignification processes from 62.9% (Table 1) to 80.9% and 79.8% in the Ck and Corg materials, while xylose proportion decreased from 24.6% (Table 1) to 18.8% and 17.4% respectively. Arabinose, galactose and acetyl groups are no longer present in both pulped cardoon samples, while mannose proportion only decreased for the kraft pulp.

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The steam exploded samples showed in both cases a decrease of the xylose proportion. In the case of CSE-k glucose proportion increased while CSE-org presented acetyl groups and similar glucose proportion.

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Py-GC/MS analysis

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The lignin-derived compounds obtained by pyrolysis of all the samples (C, CSE, Ck, Corg, CSE-k and CSE-org) are presented in Table 3, and quantified as relative molar abundances and presented as % of the identified compounds.

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The lignin-derived compounds are from syringyl (S) and guaiacyl units (G), and no hydroxyphenyl units (H) were detected. The most important compounds were 48 ACS Paragon Plus Environment

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vinylsyringol (18), trans-4-propenylsyringol (21), 4-vinylguaiacol (4), 4-methylsyringol (9), and syringol (6), and guaiacol (1). The S/G ratio of these samples was similar, respectively 1.35 and 1.40 for C and CSE.

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The kraft delignified samples presented somewhat more guaiacyl-derived compounds and a slightly lower S/G ratio of 1.25 for Ck and 1.23 for CSE-K in comparison to the original samples.

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The organosolv treated samples had a lignin composition not far to the starting material with a S/G of 1.54 and 1.47 for respectively Corg and CSE-org.

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Discussion

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Chemical characterization of cardoon

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The chemical characterization of the cardoon sample used in this work (Table 1) is in the range of values reported in the literature14,15,24,29. Ash content is within the range of 4.6-9.0%14,15,24,30.

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The extractives content was similar to the values given for Cynara stalks of 11.2%16, 14.3%30 and 14.6 %21, but higher than the 8.9% reported by Lourenço et al.24. The high extractives content was mainly due to the water and ethanol soluble compounds, respectively 52.8% and 43.6% of the total extractives. The lipophilic extracts represented only a minor part of the extractives (3.6%) as it is common for cardoon24,31.

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Total lignin content was similar to the reported range of 19.9% to 21.5 %21,24,29 and with a composition as regards S/G ratio (Table 3) similar to reported values (0.7, 1.3 and 2.124,25.

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The neutral sugars composition was also near to that reported by other authors15,24,29. Cardoon hemicelluloses are mainly xylans i.e. xylose represented 24.6% of the total monomers with a substantial acetyl substitution but few arabinose units (1.1%).

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The cellulose in cardoon showed a crystallinity index of 43.8% (Figure 2). This is the first time that cardoon cellulose crystallinity is reported; it is higher than the CI reported for other non-woody species such as jute (34.3%), but lower than that of sisal (57.3%)32.

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The lipophilic composition of cardoon extractives (S1) showed that the main compounds were hexadecanoic acid, β-amyrin, taraxasterol, sitosteryl-3β-Dglucopyranoside, and stigmasterol. These compounds have already been identified in cardoon and their distribution differs depending on the part of plant31. The steryl glycosides were here identified for the first time in cardoon; this type of compounds may have a negative effect during kraft pulping33. Extractives are now studied for different purposes, namely related to their potential bioactivity, and it is known that several pentacyclic triterpenes, sterols, aromatics and fatty acids have valuable medicinal properties34-36. However, the content of cardoon stalks in such components is small (Table 1) which may hinder their potential valorization.

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Steam explosion 9 ACS Paragon Plus Environment


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The steam explosion induced a loss of 12% of the initial solid material that did not spread homogeneously across all the chemical components of cardoon. When comparing the chemical composition of CSE with the initial cardoon (Table 1), it can be seen that steam explosion induced a selectively higher removal of inorganic materials and of extractives while lignin content remained approximately constant.

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Under the tested conditions, steam explosion did not selectively remove lignin (only about 7% of the cardoon lignin were removed) nor promoted lignin recondensation as obtained by Fernandes et al.15 who reported a higher Klason lignin content (37.7 %) after steam explosion. Negro et al.5 also reported that Klason lignin can increase in steam exploded samples by formation of pseudolignins. Steam explosion neither did alter the lignin composition and the S/G ratio of CSE was similar to that of the original material (1.40 vs. 1.35, Table 3). Again this is not as discussed in the literature, where steam explosion is said to induce modifications on the lignin structure37 with a decrease of methoxyl groups due to demethoxylation reactions and degradation of syringyl units15, leading to a S/G ratio reduction in steam exploded samples4.

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The steam explosion conditions were chosen to prevent a large mass loss, with removal of non-structural components and less of structural components. The results obtained are consistent with this idea. In fact, the effect of steam explosion depends on the applied severity, e.g. higher temperature and/or treatment time will increase mass loss and chemical changes. For instance, Fernandes et al.15 treated cardoon with a higher severity factor of 3.97 and obtained a lower solid yield (75.8% vs. 88% obtained here), and higher chemical differences. Also Arias17 applied a range of severity factors from 2.6 to 5.0, and had more chemical changes with the higher severity conditions, namely a decrease of lignin content to 16.3%.

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An effect of the steam explosion was indeed found on the relative proportion of polysaccharides (Table 1). Holocellulose decreased and α-cellulose increased in the steam exploded cardoon, and there was a selective removal of the hemicellulosic monosaccharides i.e. all sugars relative proportion decreased except glucose. This is line with the higher reactivity of hemicelluloses that are easily hydrolysed to oligo- or monosaccharides depending on the intensity of the steam explosion treatment37. The solubilization of arabinose is a common feature during steam explosion38. Other biomass pre-treatments have shown that hemicelluloses are more easily depolymerized while lignin is more resistant: for instance, the hydrothermal treatment (autohydrolysis) of corn straw treated at a similar severity factor of 3.75 depolymerized 72% of the original xylan while lignin and cellulose were not affected39; similar results were obtained in the autohydrolysis of rice straw40.

379 380 381 382

Cellulose in the steam-exploded cardoon increased to a small extent its crystallinity index to 47.9% (Figure 3). This can be explained by the selective removal of the cellulose chains not associated with the crystalline lattice, i.e. the treatment only acted upon the amorphous cellulose41.

383 384 385 386 387 388

The visual effect of steam explosion on cardoon (Figure 1) was an increase in the particle size homogeneity by reducing the large fibrous clusters, certainly resulting from the physical effects of pressuring and depressurizing and an overall darkening (∆L* = 23). Negro et al.5 also referred the darkening of pine chips after steam explosion that is attributed to the occurring chemical changes e.g. loss of hemicelluloses and sugar degradation, lignin breakdown and condensation of extractives9. 10 ACS Paragon Plus Environment

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389 390 391 392 393

Steam explosion induced some changes in the composition of the lipophilic extractives (S1, Figure 3, Figure 4). Steryl glycosides were distinctively lower in CSE comparatively to C, while fatty acids were higher and triterpenes lower (Figure 3). The literature is scarce on the influence of extractives composition after steam explosion but they should undergo hydrolysis and rearrangements that may explain the differences found here.

394

Delignification

395 396 397 398 399 400 401 402 403 404

The kraft process allowed to selectively remove lignin from the cardoon and steam exploded cardoon: the residual lignin was 10.4 and 10.7% respectively (Table 2) which represents a similar removal of 74.7% and 72.2% of the lignin present in the original materials. The possibility of obtaining kraft pulps from cardoon stalks had already been reported, with pulp yields ranging from 33.9% to 47.0% and Kappa numbers from 11 to 20.8 (which represents approximately 1.4 to 2.7% of residual lignin in the pulps) depending on starting material and reaction conditions (20-25% alkali, 30% sulfidity, 160-170ºC)21,42. In relation to such pulping experiments, the reaction conditions used in the present work were milder e.g. 18% alkali, thereby explaining the still relatively high residual lignin in the pulps.

405 406 407 408 409

No significant differences were found in the delignification of the steam exploded cardoon in comparison with the untreated sample (Table 2) except for the higher yield (50.9% vs. 45.1%). In kraft delignification the lignin β–O–4´ linkages are easily attacked43 as the syringyl units are more reactive44. Therefore, the S/G ratio slightly decreased in the pulped C and CSE samples (1.25 and 1.23 respectively, Table 3).

410 411 412 413 414 415 416 417 418

The results obtained with the organosolv treatment were quite different. In fact the ethanol:water medium was unable to selectively remove lignin i.e. the residual lignin was similar to the lignin content in the initial materials (21.1% for Corg and 23.0% for CSE-org, Table 3). This represented a lignin removal from the initial matrix of 30.5% in Corg and only 13.8% in CSE-org. The lignin composition was not altered in the direction of a preferential syringyl removal (Table 3). On the contrary, there was a small opposite trend with some decrease of guaiacyl units (the S/G ratios slightly increased to 1.54 and 1.47 in Corg and CSE-org respectively). This may be related with a differential removal of guaiacyl units linked to hemicelluloses44.

419 420 421 422 423 424 425 426 427 428 429

In fact, the organosolv conditions used were too mild (33% ethanol, 160ºC) and the acid catalysis (0.1% H2SO4) not suitable for a strong selective lignin removal. Shatalov and Pereira45 needed harsh reaction conditions of 35% alkali addition to obtain well delignified organosolv pulps from cardoon. Although ethanol:water pulping has been reported as an alternative method to obtain pulps, the reaction conditions are important to determine the extent of delignification. For instance, Pereira et al.46 reported a lignin removal of almost 70% from eucalypt chips at a pulp yield of 60% at 175ºC, but at 160ºC with 1 h reaction only 36% of lignin was extracted which is comparable to the present results. However, pre-treatments may influence the organosolv reactions and Moniz et al.47 could remove about 40% of the lignin of auto-hydrolysed rice straw with a very mild organosolv treatment at 30 ºC.

430 431 432

The steam explosion treatment did not enhance the delignification, in fact the lignin removal was even slightly smaller in the steam exploded cardoon, probably due to some condensation reactions that occurred during the treatment48. 11 ACS Paragon Plus Environment


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433 434 435 436 437 438 439 440 441 442 443 444 445 446 447

These results show that steam explosion of cardoon biomass at the mild conditions applied here had no significant effect upon subsequent lignin removal i.e. when the steam exploded material was delignified no great advantage was obtained. This is against the idea that steam explosion allows higher defibration without degradation of the fibers15, that can contribute for an easier delignification because of the structure loosening and better diffusion of the cooking liquor into the fibers49. Instead the present results stress that the effect of steam explosion is influenced by the combination of temperature, residence time and particle size and a severity factor above 3.14 is needed for an impact on a subsequent delignification. For instance, Ballesteros et al.9 was able to dissolve more lignin with soda pulping when the steam explosion was performed with high severity at 210 ºC and 8 min. The use of an unfractionated triturated sample of cardoon that included different particle sizes (Figure 1), which would be a requisite to maintain a low level in raw material cost preparation, also contributes to explain the low lignin removal by steam explosion and its inefficacity as a selective delignification pre-treatment.

448 449 450 451

In summary, the steam explosion is a clean and cost effective biomass pre-treatment that removes inorganic material, extractives and hemicelluloses in the liquid stream, thereby concentrating the solid in cellulose and lignin for which both options of lignin removal or polysaccharide hydrolysis may be considered as matrix fractionation routes.

452 453 454

AUTHOR INFORMATION

455

Corresponding Author

456 457 458

* Ana Lourenço; [email protected]; Centro de Estudos Florestais, Instituto Superior de Agronomia, Universidade de Lisboa, Tapada da Ajuda, 1349-017 Lisboa, Portugal. Phone: +351213653384; fax: +351213653338.

459 460

Author Contributions

461 462 463 464 465

The work was planned by AL, JG, HP, ER and JCV, and the material was provided by MDC. AL and ER prepared the material, preformed the steam explosion treatment and produced the pulps. AL performed the GC/MS, Py-GC/MS analysis, compiled the results of all the analysis performed and wrote the article with collaboration of the other authors. All authors have given approval to the final version of the manuscript.

466 467

Funding Sources

468 469

The first author thanks the Cost Action for a Short Term Scientific Mission (COSTSTSM-FP1203-21567) and Fundação para a Ciência e a Tecnologia (FCT) for a post12 ACS Paragon Plus Environment

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470 471

doctoral grant (SFRH/BPD/95385/2013). The research was financed by FCT through the base funding of the Forest Research Center (CEF) under UID/AGR/00239/2013.

472 473 474

Acknowledgments

475 476

We thank Duarte Neiva and Catarina Chemetova for the chemical analysis of the cardoon samples.

477 478 479

"Supporting Information. **List of identified DCM extractives present on cardoon and cardoon after steam explosion.**"

480 481 482

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Tables Table 1. Chemical characterization of cardoon (C) and cardoon after steam explosion treatment (CSE). Mean values of two replicates. C Ash (% o.d. material)

CSE

7.4

5.3

14.0 0.5

12.4 0.6

Ethanol

6.1

5.3

Water

7.4

6.5

Klason lignin

18.6 16.1

19.6 17.4

Soluble lignin

2.5

2.1

Holocellulose (% o.d. material)

74.3

68.4

α-cellulose (% o.d. material)

47.3

52.2

1.1

< 0.4

24.6

23.4

Mannose

2.6

2.2

Galactose

1.9

1.0

62.9

68.0

7.0

5.0

Total extractives (% o.d. material) Dichloromethane

Total lignin (% o.d. material)

Monosaccharides (% total neutral monosaccharides) Arabinose Xylose

Glucose Acetic acid



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 23

Table 2. Total yield (%) of the pulps produced by kraft (Ck and CSE-k) and organosolv processes (Corg and CSE-org) and their chemical characterization: total residual lignin (% of oven dry residue) and monossacharides composition (% of total neutral monosaccharides and acetic acid). Ck

CSE-k

Corg

CSE-org

Total yield (% o.d. material)

45.1

50.9

61.2

73.3

Total residual lignin (% o.d. pulp) Klason lignin

10.4 9.3

10.7 9.6

21.1 19.1

23.0 21.1

Soluble lignin

1.2

1.1

2.0

1.9

Monosaccharides (% of total neutral monosaccharides and acetic acid) Arabinose

-

-

-

-

Xylose

18.8

16.1

17.4

15.7

Mannose

0.3

0.5

2.7

2.6

Galactose

-

-

-

0.9

80.9

83.4

79.8

77.1

-

-

-

3.7

Glucose Acetic acid


Page 19 of 23

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Table 3. Identities and relative molar abundances (% of identified lignin compounds) of the lignin-derived compounds from pyrolysis of cardoon (C) and cardoon after steam explosion (CSE) and their respective delignified samples (Ck, Corg, CSE-k, CSE-org). Peak

Compound

Origin

C

Ck

COrg

CSE

CSE-k

CSE-org

1

guaiacol

G

3.5

7.1

3.0

3.3

6.7

4.3

2

4-methylguaiacol

G

4.7

5.3

4.6

4.7

5.6

5.7

3

4-ethylguaiacol

G

0.6

1.0

0.5

0.7

0.9

0.7

4

4-vinylguaiacol

G

8.6

9.0

7.4

8.1

9.8

7.8

5

eugenol

G

1.7

1.4

1.5

1.6

1.5

1.5

6

syringol

G

6.3

9.6

7.8

7.4

9.6

8.5

7 8

cis-isoeugenol trans-Isoeugenol

S G

1.5 5.2

1.7 6.2

1.0 4.4

1.6 5.2

1.5 4.8

1.3 5.2

9

4-methylsyringol

G

6.5

9.9

5.7

6.4

5.9

8.1

10

vanillin

S

3.9

5.0

4.2

3.7

4.2

3.9

11

homovanillin

G

2.2

2.7

2.5

2.0

3.0

2.2

12

4-ethylsyringol

G

1.1

1.3

1.0

0.9

1.1

1.0

13

acetoguaiacone

G

2.0

2.0

2.1

1.8

2.2

1.9

14

4-vinylsyringol

S

10.4

6.3

8.9

9.2

6.5

7.8

15

guaiacylacetone

G

1.0

1.2

0.8

0.9

1.2

1.0

16

4-allylsyringol

G

2.9

2.1

2.6

2.9

1.8

3.0

17

propioguaiacone

G

0.9

0.6

0.9

0.8

0.6

0.8

18

guaiacyl vinyl ketone

S

0.9

0.6

0.9

0.8

0.6

0.8

19 20

cis-4-Propenylsyringol 4-propinylsyringol

S G

1.8 2.1

2.3 0.9

2.1 2.8

1.8 1.9

2.5 1.4

2.0 1.9

21

trans-4-propenylsyringol

S

9.0

11.2

9.6

10.3

11.5

10.6

22

syringaldehyde

S

5.2

2.4

6.2

4.9

3.1

5.0

23

homosyringaldehyde

S

1.7

1.6

2.8

1.7

1.9

2.1

24

acetosyringone

S

2.2

2.7

2.9

3.0

4.8

3.1

25

trans-coniferyl alcohol

S

3.3

-

2.9

3.6

-

2.1

26

trans-coniferaldehyde

S

3.0

0.8

2.7

2.9

2.2

1.3

27

syringylacetone

S

1.6

2.0

1.7

1.7

1.9

1.5

28

propiosyringone

G

0.4

-

-

0.4

-

0.4

29

syringyl vinyl ketone

G

0.4

-

0.5

0.3

-

0.5

30

trans-sinapyl alcohol

S

0.6

0.6

0.8

0.5

0.7

0.7

31

trans-sinapaldehyde

S

5.1

2.5

5.3

4.8

2.5

3.3

S/G ratio 1.35 G – guaiacyl lignin unit; S – syringyl lignin unit.

1.25

1.54

1.40

1.23

1.47



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 23

Figures

Cardoon

Steam exploded cardoon

∆

L*

74.2

50.9

- 23.2

a*

4.2

7.1

+ 2.9

b*

24.1

19.7

- 4.4

∆E

23.8

Figure 1. Cardoon before (left) and after steam explosion (right) and CIELab colour measurements.


Page 21 of 23

C

CSE

6000 5000 4000 3000 2000

Iam I002

Intensity (cps)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1000 0 0

10

20

30

40

50

60

2Ɵ (deg)

Intensity (cps) C CSE

Iam

2343

I002

4107

Iam I002

2958 5260

Crystallinity Index (%) 43.8 47.9

Figure 2. X-ray diffractograms of cardoon (C) and cardoon after steam explosion (CSE) and the respective crystallinity index.



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Page 22 of 23

22

92

C

75

91 83

29 14 18

36

24

42

44 47

84 85

53 55

60 63

90

87

71

93

27 29

75

92

83 85

73 79 36

14 18

5

94

28

22

CSE

73 79

10

15

20

44

47

25

53 55

30

91 84

71

35

40

45

50

55

Time (min)

Figure 3. GC-MS chromatogram of the TMS derivatized dichloromethane extract of cardoon (C) and cardoon after steam explosion (CSE). 14 – tetradecanoic acid; 18 – pentadecanoic acid; 22 – hexadecanoic acid; 24 – heptadecanoic acid; 27 – (9Z,12Z)-Octadeca-9,12-dienoic acid; 28 – (Z)-Octadec-9-enoic acid; 29 – octadecanoic acid; 36 – eicosanoic acid; 42 – 2Palmitoylglycerol; 44 – docosanoic acid; 47 – tricosanoic acid; 53 – tetracosanoic acid; 55 – Nonacosane; 60 – 2-hydroxytetracosanoic acid; 63 – 2-hydroxypentacosanoic acid; 64 – Untriacontrane; 71 – campesterol; 73 – stigmasterol; 75 – β-amyrin; 79 – lupeol; 81 – β-amyrin acetate; 83 – taraxasterol; 84 – lupenyl acetate; 85 – lupenyl acetate isomer; 87 – ursolic acid; 90 – campesteryl-3β-D-glucopyranoside; 91 – stigmasteryl-3β-D-glucopyranoside; 92 – sitosteryl-3β-D-glucopyranoside; 93 – β-amyrin octadecanoate; 94 – taraxasteryl palmitate.


Page 23 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Pentacyclic triterpenes Fatty acids Steryl glycosides Sterols n-Alkanes α and ω-Hydroxy fatty acids Monoglycerydes C Fatty alcohols CSE

Aromatics 0

10

20

30

40

50

60 %

Figure 4. Distribution of the families of lipophilic extractives identified in GC-MS chromatogram from cardoon and cardoon after steam explosion.


Steam Explosion as a Pretreatment of Cynara ... - ACS Publications

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