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Biomass production of four Cynara cardunculusclones and lignin composition analysis

Ana Lourenco a,*, Duarte Miranda Neiva a, Jorge Gominho a,Marıa Dolores Curt b, Jesus Fern�andez b, Ant�onio Velez Marques a,c,Helena Pereira a

a Centro de Estudos Florestais, Instituto Superior de Agronomia, Universidade de Lisboa, Tapada da Ajuda,

1349-017, Lisboa, Portugalb Escuela T�ecnica Superior de Ingenieros Agr�onomos, Universidad Polit�ecnica de Madrid, Av. Complutense s/n,

28040, Madrid, Spainc Instituto Superior de Engenharia de Lisboa, Instituto Polit�ecnico de Lisboa, Rua Conselheiro Emıdio Navarro 1,

1959-007, Lisboa, Portugal

a r t i c l e i n f o

Article history:

Received 24 November 2014

Received in revised form

3 March 2015

Accepted 9 March 2015

Available online

Keywords:

Cardoon

Lignin

Stalks

Analytical pyrolysis

Pith

* Corresponding author. Tel.: þ351 21365338E-mail address: analourenco@isa.ulisboa.

http://dx.doi.org/10.1016/j.biombioe.2015.03.00961-9534/© 2015 Published by Elsevier Ltd.

a b s t r a c t

Four Cynara cardunculus clones, two from Portugal and two from Spain were studied for

biomass production and their lignin was characterized. The clones differed in biomass

partitioning: Spanish clones produced more capitula (54.5% vs. 43.9%), and Portuguese

clones more stalks (37.2% vs. 25.6%). The heating values (HHV0) of the stalks were similar,

ranging from 17.1 to 18.4 MJ/kg. Lignin was studied by analytical pyrolysis (Py-GC/MS(FID)),

separately in depithed stalks (stalksDP) and pith. StalksDP had in average higher relative

proportions of lignin derived compounds than pith (23.9% vs. 21.8%) with slightly different

lignin monomeric composition: pith samples were richer in syringyl units as compared to

stalksDP (64% vs. 53%), with S/G ratios of 2.1 and 1.3, respectively. The H:G:S composition

was 7:40:53 in stalksDP and 7:29:64 in pith. The lignin content ranged from 18.8% to 25.5%,

enabling a differentiation between clones and provenances.

© 2015 Published by Elsevier Ltd.

1. Introduction

Cynara cardunculus L., cardoon, is a non-woody thistle plant

that has received attention as a biomass cropwith a promising

value as feedstock within a biorefinery context. Cynara car-

dunculus is an herbaceous perennial plant, with annual cycles,

well adapted to the Mediterranean climate. The annual

biomass production may reach to 14e20 tha�1 under experi-

mental conditions [1,2] although production in large scale

4; fax: þ351 213653338.pt (A. Lourenco).09

plantations under conventional agricultural practices (dry

farming) were lower e.g. 7.5 tha�1 and 9.7 tha�1 at two Portu-

guese sites [3,4]. Raccuia and Melili [5] referred that biomass

productivity depends on genotype.

At the end of the crop annual cycle, the aboveground

biomass consists on average of 40% stalks, 25% leaves and 35%

capitula, and has a low moisture content (10e15%), which is a

major advantage compared to other crops [6,7]. The stalks

chemical composition includes 5e11% ash, 13e21% extrac-

tives, 13e20% lignin and about 53% polysaccharides [6,8,9].

b i om a s s a n d b i o e n e r g y 7 6 ( 2 0 1 5 ) 8 6e9 5 87

Cardoon has been used traditionally for different purposes:

inulin can be extracted from the roots [5], cardosins for cheese

making from the capitula [10,11] and polyphenols with phar-

macological properties from the leaves [12,13]. The green

leaves may be used as food and fodder in winter [6,7]. Oil can

be extracted from the seeds, with good quality for trans-

esterification into biodiesel [6,14,15]. Cardoon biomass has

also been the focus of research for bioenergy production by

combustion, gasification or pyrolysis [16,17], or densified to

pellets for domestic heating [18]. Furthermore, fibers for pulp

and paper production were also tested after delignification

[9,19e21]. This versatility of the cardoon biomass as a feed-

stock for different purposes potentiates this crop as a prom-

ising raw-material for biorefineries.

In this context, lignin is an important chemical component

that requires an adequate valorizationwithin the full resource

use concept. Lignin influences the biomass heating value and

may be used for the production of bio-based products e.g.

antioxidants, resins, polymers, while its content and compo-

sition influence fractionation and recovery processes [22,23].

The lignin composition in Cynara biomass has not yet been

studied, as far as we know. Therefore, the objective of this

work was to characterize the lignin in the stalks of four clones

of Cynara cardunculus by analytical pyrolysis. The variability

between the clones was also evaluated in relation to produc-

tion, biomass partitioning and lignin characterization.

2. Material and methods

2.1. Plant material

Plant material used in this work came from a field located at

the experimental farm of the Polytechnical University of

Madrid, in Spain (40�2404000N, 03�4004100W, 667 m a.s.l.), with

mean annual precipitation and temperature of 436 mm and

14.6 �C, respectively. Four cardoon plots were established in

June 2008 frommicro-propagated plants from four clones (one

plot per clone, each plot 45 m2). The mother plants were

selected from two demonstrative plantations grown in the

Iberian Peninsula, one from Portugal (plant codes P1 and P2)

and the other from Spain (plant codes E1 and E4) [24], that

were established from seeds of Cynara cardunculus L. var. altilis

(cultivated cardoon [25]). A large amount of phenotypic di-

versity can be found in cardoon crops grown from seeds, due

to the allogamous nature of this species.

After plant establishment, the crop was maintained under

rainfed conditions, following a perennial cultivation system

with annual harvests of the aboveground biomass [26].

2.2. Production of micro-propagated plants

The production ofmicro-propagated plants was carried out by

culture of shoot apices excised from actively growing lateral

buds of each mother plant. The procedure consisted of three

main stages: (i) establishment of the aseptic culture, (ii)

multiplication of propagula by repeated subcultures on

different culture media; (iii) preparation of the plantlets for

establishment in the soil, as described by Murashige [27]. It is

well-known that many factors influence the success of tissue

culture, like the composition of the culture medium and the

environmental conditions. In the present work the method-

ology described by Pe~na-Iglesias and Ayuso [28] for globe

artichoke micro-propagation was tested for cardoon and

modified accordingly.

2.3. Sampling and biomass partitioning

The plants collected for this study were harvested in their 4th

growth cycle. In the experimental site, the growing cycle of

Cynara usually extends from September (sprouting) to August

(summertime), when Cynara aboveground biomass dries up.

The best harvest time for collection of the solid biomass is at

the end of summer, when the water content (on a dry mass

basis) of the whole biomass is <15% [26]. Therefore, the

aboveground biomass produced by single clonal plants was

cut down in August by hand, leaving approx. 5 cm of stalks in

the ground. Two well-developed plants per clone were sepa-

rately taken to the laboratory and evaluated for the following

biometric traits: plant height, number of stems, biomass

weight and biomass partitioning into stems (stalks), leaves

(basal þ cauline leaves) and heads. At the same time, whole

fractions of stalks, leaves and heads of clonal plants were

evaluated for moisture content so that biomass production

and biomass partitioning could be expressed on dry matter

basis. The mean moisture content was 7%, a value consistent

with the high temperatures recorded in that month (26.9 �Cmean monthly temperature, 40.6 �C maximum temperature)

and the absence of rainfall. After biometricmeasurements the

stalks were separately placed inside closed PE bags and taken

to the laboratory for characterization.

2.4. Calorific values

The calorific values of the stalks were determined in com-

posite stalk samples of each clone. Stalks were hand-cut into

small pieces and mechanically ground in two steps to pass a

1 mm sieve and thoroughly homogenized to ensure repre-

sentative samples for energy determinations. Stalks energy

content was measured as the higher heating value (HHV0) in

moisture-free samples by calorimetry (Leco AC 3500 calorim-

eter), according to UNE-EN 14918. All determinations were

performed in triplicate.

2.5. Py-GC/MS(FID) analysis

The stalks of each plant were fractionated by hand in stalks

without pith (StalksDP) and pith (P). The samples were

extracted with a solvent sequence of dichloromethane,

ethanol and water, and milled to powder, as described else-

where [29]. The sampleswere pyrolysed in a CDS platinumcoil

Pyroprobe 2000 apparatus with a CDS 1500 valved interface

coupled to a split/splitless injector of a GC-MS/FID Thermo

Trace Ultra Polaris Ion Trap apparatus (Thermo Finnigan,

Austin, TX) equipped with a fused-silica capillary column DB-

17HT (30 m � 0.25 mm � 0.15 mm).

A 80 ± 5 mg sample was pyrolysed in a quartz boat at a

temperature of 899K for 10 swith a temperature rise timeof ca.

294Kms�1with the interfacekept at 544K. TheGC injector, FID

detector and GC-MS interface temperatures were 514 K, 524 K

b i om a s s a n d b i o e n e r g y 7 6 ( 2 0 1 5 ) 8 6e9 588

and 524 K, respectively. The GC oven programwas: 324 K, held

for 3 min, 280 K min�1e354 K, 278 K min�1e424 K,

279 K min�1e494 K, 281 K min�1e574 K, held for 10 min. For

mass spectra analysis the conditionswere: the electron impact

ionization was 70 eV, 494 K for ion source temperature and

300 mm3 min�1 of damping helium gas.

The total area of the pyrogram was obtained by automatic

integration (Thermo Excalibur software) and manually cor-

rected in some peaks for all the samples.

Identification and quantification of pyrolysis products

were performed separately by Py-GC/MS and Py-GC/FID

analysis, respectively. For identification of analytes, NIST

Mass Spectral Library (2001) and Faix et al. [30] were used. For

quantification, the relative areas to total chromatogram area

(except gases) were used. The lignin derived compounds were

classified in syringyl (S), guaiacyl (G) and p-hydroxyphenyl (H)

lignin monomeric units and their proportions calculated as

well as the S/G and H/S/G ratios.

3. Results and discussion

3.1. Biomass production and partitioning

The aboveground biomass production ranged on a single plant

basis from 1.386 kg to 2.805 kg (mean value of 2.094 kg),

measured on a dry basis (Table 1). These values are higher

than those obtained by the 0.336e0.573 kg reported for the

original fields where the mother plants were collected [4,31],

and the 1.479 kg reported by Ierna and Mauromicale [32]. This

result could be related to the origin of the plants since selected

clones were taken, and to the age of the studied plants,

because sampling was made at the 4th growing cycle.

Portuguese clones produced taller plants (1.65 me1.79 m),

and more stems per plant (5e8 stems, Table 1), comparatively

to the Spanish clones, where the plant height ranged from

1.45 m to 1.63 m and produced in average only 3 stems per

plant. A good correlation was found between the height of

plants and the number of stems (r ¼ 0.84). Previous records

reported plants with an average of 2.4 m, and 6.5 capitula [4].

Aboveground biomass partitioning is presented in Table 1:

the heads were the main fraction representing in average

49.2% of total biomass, followed by the stalks with 31.4% and

the leaves with 19.4%. The Spanish clones (E1 and E4) showed

Table 1 e Biomass production and partitioning of Cynara carduprovenances.

Clone Plant Plant height (m) N� stems/plant

E1 1 1.45 2

2 1.45 4

E4 3 1.63 3

4 1.48 3

P1 5 1.70 5

6 1.65 5

P2 7 1.73 6

8 1.79 8

Average ± STDEV 1.61 ± 0.1 4.5 ± 1.9

a higher production of heads, varying from 50.3% to 58.0%

with mean value of 54.5%, in comparison with the 43.9% for

the Portuguese clones. On the other hand, the clones from

Portugal (P1 and P2) showed better aptitude for stalk produc-

tion which represented a higher proportion of the above-

ground biomass, in average 37.2% vs. 25.6%. There were no

significant differences between the clones in respect to pro-

portion of leaves. Gominho et al. [4] without accounting the

leaves, reported 58.7% for the stalks and 41.3% for the heads.

3.2. Calorific values

The calorific values of the stalks were similar among the

studied clones, with average values of 17.6 MJ kg�1 HHV0

varying from 17.1 to 18.4 MJ kg�1. These values are higher

when compared to the 14.8 MJ kg�1 obtained from cardoon

pellets (10.9% moisture content [18]) and in the range of the

18.2 MJ kg�1 mentioned by Encinar et al. [14], but lower than

the 21.5 MJ kg�1 obtained with cardoon stems [33]. The calo-

rific value of cardoon is similar to the 16.7 MJ kg�1 and

16.9 MJ kg�1 obtained for giant reed and miscanthus [34].

Although cardoon has a high calorific value, its use for energy

production by combustion is difficult due to the low density

and the significant quantities of undesirable components (e.g.

ash, 5.4%) and should therefore be mixed with other low ash

content feedstock, as discussed by Abelha et al. [17] and

Gonz�alez et al. [18].

3.3. Cardoon pyrolysis

Fig. 1 represents an example of the Py-GC/FID chromatograms

obtained after pyrolysis of stalksDP and pith, and Table 2

presents the list of compounds identified, describing their

origin, retention times and mean value (as % of the total area

of the chromatogram).

A total of 110 compounds were identified representing 57%

of the total peak area (59.0% in stalks and 55.6% in pith sam-

ples). This value is somewhat lower to the usually reported for

wood pyrolysis analysis, e.g. Rodrigues et al. [35] reported 78%

of area identified. This can be explained by a lower identifi-

cation of the carbohydrates derived compounds, in particular

those with low molecular weight (hydroxyacetaldehyde,

hydroxypropanone, or furan derivatives). The carbohydrates

are fragmented into low molecular weight compounds, and

nculus plants from clones of Spanish and Portuguese

Biomass partitioning (%)

Dry weight(g plant�1)

Stems Leaves Heads

1572.3 24.1 22.5 53.4

2804.7 21.8 21.9 56.3

1386.3 33.9 15.8 50.3

1886.6 22.7 19.3 58.0

2713.3 29.8 18.3 51.9

1996.5 35.2 17.2 47.6

2405.4 42.5 21.5 36.0

1985.1 41.4 18.5 40.1

2093.8 ± 510.3 31.4 ± 8.2 19.4 ± 2.4 49.2 ± 7.7

Fig. 1 e Py-GC/FID chromatograms of depithed stalks (StalksDP) and pith from plant 1 of Cynara cardunculus.

b i om a s s a n d b i o e n e r g y 7 6 ( 2 0 1 5 ) 8 6e9 5 89

their origin is difficult to distinguish bymass spectrometry, as

discussed by Faix et al. [36]. Contrary, the lignin derivatives are

easier to identify since lignin pyrolysis produces a mixture of

phenol derivatives that preserve the aromatic ring and the

original methoxyl groups [37,38].

Overall, no major differences were found between the

chromatograms of stalksDP and pith regarding the peak

assignment, and only minor differences were noticed at

quantitative level (Table 2). For example, the proportion of

coniferyl alcohol (trans) was higher in stalksDP (1.6%)

comparatively to pith (0.39%). The same occurred for other

compounds: 4-vinylguaiacol with respectively, 0.95% and

0.35%, dimethoxyphenol isomer with 0.22% and 0.08%, iso-

eugenol (trans) with 0.65% and 0.20%, and acetoguaiacone

with 0.25% and 0.08%. Contrarily, 4-vinylsyringol was lower in

stalksDP (2.5%) comparatively to pith (3.4%), 4-

propenylsyringol (trans) with respectively, 1.7% and 2.3%,

syringol with 0.79% and 1.3% (Table 2).

The main lignin derived products obtained from cardoon

pyrolysis were from syringyl units (Table 2): 4-vinylsyringol

(in average 2.9% of total area), 4-propenylsyringol (2.0%),

sinapinaldehyde (1.4%), sinapyl alcohol (trans) (1.3%), syringol

(1.0%), syringaldehyde (0.9%). The sum of all syringyl com-

pounds represented from 50% to 65% of total lignin-derived

cardoon products, as discussed further. The guaiacyl units

were less abundant, varying from 28% to 42% and the

main compounds were: coniferyl alcohol (trans) (1.0% of

total area), 4-methylguaiacol (0.9%), 4-propylguaiacol (0.8%),

4-vinylguaiacol (0.7%), guaiacol (0.5%), 4-ethylguaiacol (0.4%),

isoeugenol (trans) (0.4%).

The main compound produced from carbohydrates pyrol-

ysis was levoglucosan (7.6% of total area), followed by 4-

hydroxy-5,6-dihydro-2H-pyran-2-one (3.6%), 2-furaldehyde

(2.3%), 2-hydroxymethyl-5-hydroxy-2,3-dihydro-4H-pyran-4-

one (1.5%), 3H-pyran-2,6-dione (1.3%). Typical carbohydrate-

pyrolysis compounds are furans, lactones (furanones), cyclic

ketones (cyclopentanones) as well as anhydrosugars [39].

Compounds such as hydroxyacetaldehyde, acetic acid,

hydroxypropanone, and 2-oxopropanoic acid methyl ester

that are produced during pyrolysis of miscanthus and wheat

straw [40], Lolium and Festuca grasses [41] or elephant grass

[42], were not identified in cardoon pyrolysis.

In residual traces were also found compounds defined as

UPO (undetermined phenolic origin) with the backbone of

benzenediol or toluene, that represented less than 3% of total

area of the chromatogram.

3.4. Lignin content and composition

The lignin-derived peaks represented in average 22.9% of the

total area of the chromatogram. This value is similar to the

lignin content of the whole stalk, 21.5% (on an extractive-free

basis) determined bywet chemistry [8]. On the other hand, the

lignin content of stalksDP was in average 23.9%, varying from

22.7% to 25.5%, and in pith samples the average value was

lower (21.8%), but the amplitude of variation was higher

Table 2 e Identification and quantification of the Cynara cardunculus pyrolysis products (% of total chromatographic area). Average ± STDEV of eight plants.

Peak no. Retention time (min) Compound Origin* StalksDP Pith

1 5.76 5H-furan-2-one C 0.56 ± 0.1 0.54 ± 0.1

2 6.14 Furfural isomer C 0.68 ± 0.2 0.59 ± 0.2

3 6.28 3-Buten-2-ol C 1.1 ± 0.3 1.6 ± 1.0

4 6.67 2-Furaldehyde (furfural, FF) C 2.2 ± 0.2 2.3 ± 0.2

5 7.46 5-Methyl-3H-furan-2-one (a-angelicalactone) C 0.16 ± 0.02 0.17 ± 0.02

6 8.30 2-Acetylfuran C 0.43 ± 0.1 0.51 ± 0.1

7 9. 53 Benzaldehyde H 0.05 ± 0.01 0.06 ± 0.01

8 9.64 Phenol H 0.24 ± 0.1 0.25 ± 0.1

9 11.10 3H-Pyran-2,6-dione C 1.5 ± 0.1 1.2 ± 0.7

10 11.39 4-Hydroxy-5,6-dihydro-2H-pyran-2-one C 4.1 ± 0.2 3.1 ± 0.7

11 11.50 Methyl-dihydro-(2H)-pyran-2-one C 0.87 ± 0.1 1.2 ± 0.4

12 11.71 o-Cresol H 0.23 ± 0.1 0.26 ± 0.1

13 11.91 2-Hydroxy-1-methyl-1-cyclopentene-3-one C 1.3 ± 0.2 1.1 ± 0.2

14 12.28 p-Cresol H 0.18 ± 0.03 0.20 ± 0.1

15 12.54 Dimethylanisole G 0.02 ± 0.02 0.03 ± 0.2

16 12.61 Sugar derivative (MW 112 or 114) C 0.60 ± 0.03 0.61 ± 0.1

17 13. 34 Guaiacol G 0.64 ± 0.1 0.40 ± 0.1

18 13.50 Sugar derivative (MW 114) C 0.35 ± 0.03 0.33 ± 0.04

19 13.75 1-(2-Furangyl)-2-hydroxy-ethanone C 0.13 ± 0.03 0.15 ± 0.03

20 13.80 2,3-Dihydro-5-hydroxy-6-methyl-4H-pyran-4-one C 0.26 ± 0.02 0.28 ± 0.03

21 14.01 5-Hydroxymethyl-2-furaldehyde isomer C 0.24 ± 0.04 0.34 ± 0.1

22 14.10 Sugar derivative (MW 126 or 130) C 0.32 ± 0.1 0.37 ± 0.2

23 14.19 Sugar derivative (MW 126) C 0.38 ± 0.04 0.42 ± 0.1

24 14.29 2,6-Dimethylphenol H 0.23 ± 0.01 0.21 ± 0.03

25 14.41 2,4-Dimethylphenol or Dimethylphenol isomer H 0.07 ± 0.01 0.07 ± 0.02

26 14.87 3-Hydroxy-2-methyl-4H-pyran-4-one (maltol) C 0.41 ± 0.05 0.47 ± 0.1

27 14.94 2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one C 0.31 ± 0.03 0.26 ± 0.05

28 15.08 Dimethylphenol isomer H 0.18 ± 0.02 0.18 ± 0.03

29# 15.15 5-Hydroxy-2-methyl-4H-pyran-4-one isomer/5-hydroxymethyl-2-furaldehyde isomer C 0.12 ± 0.01 0.10 ± 0.02

30 15.55 Methylguaiacol isomer G 0.04 ± 0.01 0.04 ± 0.02

31# 15.76 4-Methylguaiacol isomer/3,5-dihydroxy-2-methyl-4H-pyran-4-one isomer G/C 0.12 ± 0.03 0.11 ± 0.03

32 15.94 4-Methylguaiacol G 0.79 ± 0.1 1.1 ± 0.4

33,34 16.04,16.13 3,5-Dihydroxy-2-methyl-4H-pyran-4-one isomer C 0.41 ± 0.1 0.42 ± 0.03

35 16.31 2H-Pyran-2-one C 0.65 ± 0.1 0.74 ± 0.1

36 16.40 1,2-Benzenediol (catechol) or resorcinol UPO 0.13 ± 0.04 0.16 ± 0.04

37 18.36 Dimethoxytoluene isomer UPO 0.08 ± 0.04 0.08 ± 0.03

38 18.71 4-Ethylguaiacol G 0.37 ± 0.1 0.35 ± 0.1

39 18.87 3-Methyl-1,2-benzenediol UPO 0.29 ± 0.1 0.29 ± 0.04

40 19.08 a-b-D-Ribopyranose-1,3-di-O-acetyl C 0.52 ± 0.2 0.61 ± 0.1

41 19.20 1,4-3,6-Dianhydro-glucopyranose C 0.35 ± 0.1 0.46 ± 0.1

42 19.34 3-Methoxy-1,2-benzenediol UPO 0.40 ± 0.1 0.44 ± 0.03

43 19.46 Sugar derivative (MW 114) C 0.44 ± 0.2 0.61 ± 0.1

44# 19.70 4-Methyl-1,2-benzenediol/sugar derivative (MW 114) UPO/C 0.13 ± 0.1 0.11 ± 0.03

45 19.81 Sugar derivative (MW 114) C 0.26 ± 0.1 0.30 ± 0.1

46 19.89 5-Hydroxymethyl-2-furaldehyde C 1.2 ± 0.1 1.2 ± 0.3

47 20.06 Sugar derivative (MW 114) C 0.58 ± 0.1 0.48 ± 0.1

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mass

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bio

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76

(2015)86e95

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48 20.44 4-Vinylguaiacol G 0.95 ± 0.1 0.35 ± 0.1

49 20.96 3-Methoxy-4,5,6-trimethoxyphenol or C3-guaiacol isomer G 0.06 ± 0.02 0.08 ± 0.02

50 21.08 3-Methoxy-5-methylphenol G 0.14 ± 0.01 0.14 ± 0.05

51 21.23 4-Propylguaiacol G 0.87 ± 0.1 0.82 ± 0.2

52 21.30 Eugenol G 0.17 ± 0.1 0.16 ± 0.03

53 21.42 Dimethoxyphenol isomer S 0.22 ± 0.03 0.08 ± 0.02

54 21.57 1,5-Anhydro-arabinofuranose C 0.05 ± 0.01 0.06 ± 0.02

55 21.86 2-Hydroxymethyl-5-hydroxy-2,3-dihydro-4H-pyran-4-one C 1.7 ± 0.2 1.3 ± 0.5

56 22.16 Anhydride phtalic UPO 0.10 ± 0.01 0.11 ± 0.02

57 22.48 3,4-Dimethoxystyrene UPO 0.29 ± 0.1 0.23 ± 0.04

58 23.12 Syringol S 0.79 ± 0.1 1.3 ± 0.4

59 23.25 Isoeugenol (cis) G 0.17 ± 0.02 0.39 ± 0.1

60,69,70,72,77 23.66/27.32/27.45/28.38/29.28 Lignin derivative UPO 1.32 ± 0.3 1.21 ± 0.1

61 24.47 Isoeugenol (trans) G 0.65 ± 0.1 0.20 ± 0.1

62 24.80 Vanillin G 0.45 ± 0.1 0.17 ± 0.1

63 24.95 Methoxy-1,2-benzenediol isomer UPO 0.11 ± 0.1 0.10 ± 0.1

64 25.51 4-Methylsyringol S 0.70 ± 0.1 0.84 ± 0.1

65,66 26.24/26.41 C10H10O2 (G-C]C]C) G 0.51 ± 0.1 0.20 ± 0.04

67 26.53 Homovanillin G 0.29 ± 0.1 0.13 ± 0.02

68 27.21 Acetoguaiacone G 0.25 ± 0.04 0.08 ± 0.03

71 27.70 Methyl-4-hydroxy-3-methoxybenzoate G 0.07 ± 0.03 0.06 ± 0.02

73 28.45 Homovanillic alcohol G 0.03 ± 0.01 0.05 ± 0.02

74 28.50 Guaiacyl acetone G 0.09 ± 0.01 0.06 ± 0.02

75 28.83 1,6-Anhydro-b-D-glucopyranose (Levoglucosan, LG) C 7.7 ± 0.9 7.5 ± 3.6

76 28.91 4-Vinylsyringol S 2.5 ± 0.9 3.4 ± 1.2

78 29.37 Propioguaiacone G 0.03 ± 0.01 0.03 ± 0.02

79 29.57 4-Allylsyringol S 0.003 ± 0.003 0.02 ± 0.01

80 29.63 Coniferyl alcohol (cis) G 0.50 ± 0.02 0.76 ± 0.1

81 29.82 4-Prop-2-en-1-one-guaiacol G 0.09 ± 0.02 0.02 ± 0.005

82 29.91 Vinylsyringol isomer S 0.03 ± 0.01 0.03 ± 0.01

83 30.33 Propenylsyringol isomer S 0.10 ± 0.01 0.10 ± 0.01

84 30.95 4-Propenylsyringol (cis) S 0.02 ± 0.03 0.04 ± 0.1

85 31.09 Dihydroconiferyl alcohol G 0.06 ± 0.04 0.01 ± 0.01

86,87 31.91/32.13 C11H12O3 (SeC]C]C) S 1.0 ± 0.1 1.1 ± 0.1

88 31.98 4-Propenylsyringol (trans) S 1.7 ± 0.1 2.3 ± 0.3

89 32.40 Syringaldehyde S 0.93 ± 0.1 0.89 ± 0.1

90 32.55 Syringaldehyde isomer S 0.11 ± 0.03 0.09 ± 0.02

91 32.64 3,4-Dimethoxy-1,2-benzenediol UPO 0.07 ± 0.03 0.04 ± 0.02

92 32.83 Sugar derivative C 2.0 ± 0.4 1.6 ± 0.5

93 33.26 Homosyringaldehyde S 0.20 ± 0.05 0.22 ± 0.1

94 33.35 Coniferaldehyde G 0.76 ± 0.1 0.21 ± 0.1

95 33.50 Coniferyl alcohol (trans) G 1.6 ± 0.3 0.39 ± 0.2

96 33.69 Acetosyringone S 0.41 ± 0.04 0.35 ± 0.1

97 34.64 Syringyl acetone S 0.17 ± 0.04 0.13 ± 0.1

98 34.94 2-Propenoic acid-3-[4-acetyloxy-3-methoxyphenyl]-methylester G 0.07 ± 0.03 0.06 ± 0.03

99 35.06 Propiosyringone S 0.05 ± 0.01 0.05 ± 0.01

100 35.17 Sinapinaldehyde isomer S 0.08 ± 0.01 0.06 ± 0.02

101 35. 24 S-lignin derivative S 0.11 ± 0.03 0.10 ± 0.04

(continued on next page)

bio

mass

and

bio

energy

76

(2015)86e95

91

Table

2e

(con

tinued

)

Peakno.

Retentiontim

e(m

in)

Com

pound

Origin*

Stalks D

PPith

102

35.34

Sinapylalcohol(cis)

S0.10±0.01

0.11±0.02

103

35.47

4-Pro

p-2-en-1-one-syringol

S0.05±0.01

0.04±0.01

104

35.91

Sinapinaldehydeisomer

S0.04±0.004

0.04±0.01

105

36.44

Dihydro

sinapylalcohol

S0.06±0.01

0.04±0.01

106

36.97

Sinapylalcoholisomer

S0.42±0.06

0.30±0.1

107

37.82

Sinapinaldehyde

S1.5

±0.2

1.3

±0.2

108

38.14

Sinapylalcohol(trans)

S1.4

±0.3

1.1

±0.6

109

44.96

Cinnamic

compound(M

W276)

H0.31±0.1

0.31±0.2

110

47.64

Biphenyl(M

W302)

UPO

0.15±0.1

0.15±0.1

Totalareaiden

tified

(%totalarea)

59.0

55.6

#,Compoundsoverlapped;*C

,Carb

ohydratesderivedpro

ducts;

G,Guaiacy

lderivedunits;

S,SyringylderivedunitsandH,p-H

ydro

xyphenylderivedunits;

UPO,undeterm

inedphenolicorigin.

b i om a s s a n d b i o e n e r g y 7 6 ( 2 0 1 5 ) 8 6e9 592

(18.8%e24.6%) (Table 3). There was no statistical difference (at

a ¼ 0.05) between stalksDP and pith, or between clones as

determined by the ShapiroeWilks test. The amplitude of

values opens the opportunity for selection of clones with low

lignin content e.g, for pulp products.

The variation of lignin content between stalksDP and pith in

the mother plants was also reported by Pereira et al. [8] and

Gominho et al. [9] and can be explained by their different

anatomical features. The pith region is mainly characterized

by parenchyma cells (with thin walls), whereas the stalksDP

contain vessels and fibers (with thickened walls), as discussed

by Quilh�o et al. [43].

In all samples, the lignin monomeric composition was

mainly of syringyl-units (Table 4): in pith samples values

ranged from 62% to 65% of total lignin, while in stalksDP from

50% to 54%. Guaiacyl-units represented slightly less, 40%

average for stalksDP (varying from 39% to 42%), and 29% (28%e

31%) for pith. These results reinforce the idea that the lignin

composition differs with cell tissue, as mentioned before.

StalksDP structure presents vessels and fibers, with the first

characterized by a guaiacyl-unit type lignin and the latter by

higher abundance of syringyl-units [44], resulting in high

values of both these lignin units. Pith contains only paren-

chyma cells that were characterized with more syringyl-units

comparatively to guaiacyl-units. This is in agreement with

Chesson et al. [45] who studied the cell wall composition of

maize, and recovered more syringyl units from the paren-

chyma cells.

The H:G:S was 7:40:53 in stalksDP and 7:29:64 in pith sam-

ples (Table 4), meaning that the cardoon is mainly charac-

terized by an SG type of lignin, with more syringyl units

compared to guaiacyl, and with minor content of

hydroxyphenyl-units. This feature is similar to hardwoods

lignins (4:56:40 in beech [46]) and other herbaceous likeArundo

donax (10:43:47 [47]), but slightly different to miscanthus

where the lignin is of HGS-type (24:53:23 [48]).

Regarding the S/G ratio, the valueswere higher for pith (2.1)

when compared to stalksDP (1.3). These S/G ratio values are in

the range of the 0.34e2.19, obtained for species used for bio-

energy purposes, such as poplar, switchgrass, andmiscanthus

[48e50], and are in accordance to the 1.7 and 2.9 obtained for

other non-woody plants [51], and in the lower range of the 1.9

to 4.3 obtained for other Eucalyptus [52], in particular for

Eucalyptus globulus [29].

A high S/G ratio means that this lignin presents a more

open matrix, with a lower degree of CeC bonds at the C5-ring

position, being easier to remove during pulping [29]. There-

fore, the results obtained here confirm the potential of

cardoon for pulping, as mentioned by Gominho et al. [9].

Lignin content has been related to biomass high heating

values (HHV0) [53,54]. In the present study a high correlation

was found (r ¼ 0.97) between lignin content, calculated for the

whole stalk according to Gominho et al. [9] (i.e. pith repre-

senting 10% of stalks), and calorific values.

4. Conclusion

The production of Cynara cardunculus biomass is dominated by

the proportion of the capitula, with stalks representing nearly

Table 3 e Characterization of StalksDP and pith from Cynara cardunculus clones by Py-GC/FID. Average values of two plantsfor each clone (% of total area of the chromatogram), themean and standard deviation of all plants, and data from chemicalanalysis from references.

StalksDP

Clones Clone E1 Clone E4 Clone P1 Clone P2 Mean ± STDEV Pereira et al. [8] Gominho et al. [9]

Total extractives e e e e e 15.6 13.8

Total lignin 22.7 25.5 22.7 24.8 23.9 ± 1.5 16.1 15.8

Polysaccharides e e e e e 65.6 63.5

Total UPO* 3.0 2.7 3.1 3.1 2.9 ± 0.3

Total others** 36.6 33.4 34.7 35.0 34.9 ± 1.4

Pith

Total extractives e e e e e 13.2 12.9

Total lignin 24.6 21.7 22.2 18.8 21.8 ± 2.3 23.4 23.3

Polysaccharides e e e e e 48.0 56.6

Total UPOa 2.7 3.2 2.8 2.6 2.8 ± 0.3

Total othersb 28.9 37.4 32.2 36.4 33.7 ± 4.0

Clone E1: average of plants 1 and 2; Clone E4: average of plants 3 and 4; Clone P1: average of plants 5 and 6; Clone P2: average of plants 7 and 8.a Sum of compounds with undetermined phenolic origin (UPO).b Sum of total UPO and total carbohydrates. Refs. [8] and [9] values for stalks DP and pith not corrected to extractives content, determined bywet

chemistry. e not determined.

Table 4 e Lignin monomeric composition (% of totallignin) of StalksDP and pith of the Cynara cardunculusclones.

Stalks DP

Clones Clone E1 Clone E4 Clone P1 Clone P2 Av ± STDEV

S 50 52 54 53 53 ± 2

G 42 42 39 41 40 ± 2

H 8 6 7 7 7 ± 1

S/G 1.2 1.2 1.4 1.4 1.3 ± 0.1

Pith

S 62 65 63 65 64 ± 2

G 31 28 28 28 29 ± 2

H 7 7 9 7 7 ± 1

S/G 2.0 2.4 2.2 1.8 2.1 ± 0.3

b i om a s s a n d b i o e n e r g y 7 6 ( 2 0 1 5 ) 8 6e9 5 93

one third of the aboveground biomass at harvest. Differences

in the proportion of stalks were found between clones with

different origins, withmore stalks produced by the Portuguese

clones.

The lignin content of stalks as determined by pyrolysis was

in average 22.9% and had amonomeric composition similar to

hardwoods, consisting mainly of syringyl and guaiacyl units.

The pith and the depithed stalks showed different variation

amplitudes in lignin content: in pith the variation was higher

(18.8e24.6% vs. 22.7e25.5%); and differed in lignin composi-

tion with a higher content in syringyl units in pith.

The results suggest that there is potential to select clones

with different biomass partitioning for specifically targeted

biomass utilization: enhancement of capitula e.g. for pro-

duction of seeds for oil extraction or cardosin for cheese

making, or enhancement of stalks e.g. for fiber related uses.

Acknowledgments

This work was financed in part by the national funding of

Fundac~ao para a Ciencia e a Tecnologia (FCT) for support of

Centro de Estudos Florestais (CEF) (PEst-OE/AGR/UI0239/2014),

and by the projects PTDC/AGR-CFL/110419/2009 and PTDC/

AGR-FOR/3872/2012. The first author was funded by FCT

through a post-doctoral grant (SFRH/BPD/95385/2013).

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