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LOS DIRECTORES
EL TUTOR
Quiero expresar mi sincero agradecimiento a las personas que tanto directa como
indirectamente han hecho posible la realizacin de esta Tesis:
A los Dres. Jos Carlos del Ro y Ana Gutirrez, directores de esta Tesis, por todo
lo que me han aportado tanto a nivel cientfico, por sus conocimientos y enseanzas,
como a nivel personal, por la confianza en su trato personal y por sus consejos, y por
estar siempre que los he necesitado. Por su esfuerzo y dedicacin en esta Tesis.
A mis compaeras la Dra. Isabel Mara Rodrguez y Setefilla Molina, con las que
he coincidido en el inicio y mediados de esta Tesis, y a mis compaeros Pepijn
Prinsen, Alejandro Rico y Esteban Babot con los que he coincidido en el final de esta
Tesis. Un agradecimiento muy especial a Esteban por su apoyo y los buenos
momentos compartidos.
A Da. Trinidad Verdejo por hacer las pirlisis de mis numerosas muestras.
Al Prof. Jess Jimnez-Barbero, al Dr. Iaki Santos y a Lidia Nieto del CIB-
CSIC, por sus mltiples anlisis de NMR.
A Gerardo Artal (CELESA) por suministrarme las muestras de diversas fibras y sus
pastas, al Dr. Javier Romero (ENCE) por las pastas de eucalipto y al Dr. Manuel J.
Daz Blanco (Universidad de Huelva) por las muestras de caa comn y tagasaste.
Al Dr. Garca Hortal (UPC, Terrassa) por las imgenes proporcionadas de las
fibras elementales de las muestras de lino, camo, kenaf, yute, sisal y abac que se
muestran en la seccin de Material y Mtodos de esta Tesis.
A mis compaeros del IRNAS, Roco, Mari Trini, Agi, Alegra, Mara Fernanda,
Ftima, Jos Mara, Antonio y Jaime, que me acompaaron durante el inicio de esta
Tesis, y en particular a Ftima Sopea que tambin me acompa durante casi toda
la Tesis brindndome muy buenos momentos y consejos y por estar siempre all
incluso durante su post-doc en el extranjero.
A los compaeros del CIB, Mario, Yuta, Mara, ngeles, Miguel, Elvira, Davinia,
Vero, Helena, Aitor, Eva y Beatriz, y en particular a Celia Mndez por su apoyo en
mi primera estancia en el CIB y por los buenos momentos brindados. Quiero
agradecer tambin al Dr. Javier Ruiz-Dueas por su apoyo en las estancias
realizadas en el CIB.
A Mara Jess Ortega, madre de Aitor del CIB, aunque slo la conozco por
Internet, por sus e-mails y por proporcionarme una de las fotos que se muestra en la
seccin de Introduccin de esta Tesis.
Gracias a todos y gracias tambin, tan slo por su existencia, a una nueva
personita subacutica que ahora llevo dentro
ABREVIATURAS
AQ Antraquinona
ABTS 2,2-azinobis(3-etilbenzotiazolin-6-sulfonato)
BSTFA N,O-bis-(trimetilsilil)-trifluoroacetamida
GC Desplazamiento qumico del carbono
GH Desplazamiento qumico del protn
CED Cobre (II)-etilendiamina
COSY Espectroscopia de correlacin (Correlation Spectroscopy)
DBO Demanda biolgica de oxgeno
DCM Diclorometano
DMAC N,N-dimetilacetamida
DMSO Dimetilsulfxido
DTT Ditiotreitol
2D-NMR Espectroscopa de Resonancia Magntica Nuclear bidimensional
3D-NMR Espectroscopa de Resonancia Magntica Nuclear tridimensional
ECF Secuencia de blanqueo libre de cloro elemental (elemental
chlorine free)
FAO Organizacin de las Naciones Unidas para la Agricultura y
Alimentacin (The Food and Agriculture Organization of the
United Nations)
FID Detector de ionizacin de llama (flame ionization detector)
G Unidad guayacilpropano (o guayacilo)
GC Cromatografa de gases (gas chromatography)
GC/MS Cromatografa de gases/espectrometra de masas (gas
chromatography/mass spectrometry)
H Unidad 4-hidroxifenilpropano (o 4-hidroxifenilo)
HBT 1-Hidroxibenzotriazol
HPLC Cromatografa lquida de alta resolucin
HSQC Espectroscopa 2D de correlacin heteronuclear de cuanto simple
(heteronuclear single-quantum correlation)
HexA cidos hexenurnicos
ICP-OES Espectrometra de emisin ptica con plasma acoplado
inductivamente (inductively coupled plasma-optical emission
spectrometry)
ID Dimetro interno (internal diameter)
IK ndice Kappa
IPTG Isopropil tio--D-galactopiransido
ISO Organizacin Internacional para la Estandarizacin,
Documentacin e Informacin (International Organization for
Standardization)
ITD Detector de trampa de iones (ion trap detector)
Ligninaox Productos de degradacin oxidativa de la lignina
LiP Lignina peroxidasa
Mw Masa molecular (molecular weight)
MnP Manganeso peroxidasa
MWL Lignina de madera molida (milled wood lignin)
NMR Espectroscopa de Resonancia Magntica Nuclear (nuclear
magnetic resonance)
O Etapa de deslignificacin con oxgeno (en secuencia de blanqueo)
PCA cido p-cumrico
POM polioxometalato
POM 1- Primera etapa POM en los ensayos de deslignificacin
-POM 2- Segunda etapa POM en los ensayos de deslignificacin
POMox Polioxometalato oxidado
POMred Polioxometalato reducido
PoP Doble etapa de blanqueo con perxido de hidrgeno, la primera
bajo oxgeno presurizado
ppb Partes por billn
ppm Partes por milln
Py-GC/MS Pirlisis acoplada a cromatografa de gases/espectrometra de
masas (pyrolysis-gas chromatography/mass spectrometry)
Q Etapa de quelato (en secuencia de blanqueo)
rpm Revoluciones por minuto
S Unidad siringilpropano (o siringilo)
SPE Extraccin en fase slida (solid phase extraction)
TAPPI Technical Association of the Pulp and Paper Industry
TCF Secuencia de blanqueo totalmente libre de cloro (totally chlorine
free)
TMAH Hidrxido de tetrametilamonio
TMP Pasta termomecnica (thermomechanical pulp)
TMSD Trimetilsilildiazometano
TMS Trimetilsililo
TOCSY Espectroscopia de correlacin total (Total Correlation
Spectroscopy)
U Unidad de actividad enzimtica
UV/VIS Espectroscopa de ultravioleta/visible
VP Peroxidasa verstil
Coeficiente de extincin molar
NDICE
RESUMEN ..........................................................................................................1
1. INTRODUCCIN.........................................................................................5
2. OBJETIVOS ................................................................................................37
4. REFERENCIAS ..........................................................................................75
5. RESULTADOS Y DISCUSIN.................................................................91
6. CONCLUSIONES .....................................................................................273
7. ANEXOS ....................................................................................................277
RESUMEN
1
insaturados se eliminan durante los procesos de blanqueo ECF y TCF, los cidos
grasos saturados, as como los alcanos y alcoholes grasos sobreviven a estas
secuencias de blanqueo.
2
despolimerizacin y desacetilacin significativa durante el proceso de pasteado.
Los grupos acetilo residuales que quedaban en la pasta cruda se eliminaron
completamente tras el blanqueo.
3
1
INTRODUCCIN
Las conferas presentan fibras largas (3 a 5 mm), que son ptimas para la
fabricacin de papeles de elevada resistencia mecnica. Las conferas, en
trminos econmicos generales, son ms valiosas que las frondosas, ya que sus
troncos son ms largos y rectos, su madera es uniforme, ligera y blanda, por lo
que es ms fcil de trabajar, y presentan una mayor proporcin de elementos
5
1. Introduccin
6
1. Introduccin
Rendimiento Rendimiento
Especies materia seca (t/ha) Pasta (t/ha)
Trigo 2,5 1,1
Avena 1,6 0,7
Centeno 2,2 1,1
Arroz 3,0 1,2
Caa de azcar (bagazo) 9,0 4,2
Bamb 4,0 1,6
Miscanthus sinensis 12,0 5,7
Canary grass 6,0 3,0
Caa comn 9,0 4,3
Kenaf 15,0 6,5
Camo 12,0 6,7
Frondosa de zona templada (abedul) 3,4 1,7
Frondosa de crecimiento rpido (Eucalyptus) 15,0 7,4
Confera escandinava 1,5 0,7
Confera de crecimiento rpido 8,6 4,0
7
1. Introduccin
Lino Yute
Papel para bolsas de vaco
Bolsas de t
Camo Sisal
Papeles para filtros
Papeles
decorativos
Kenaf Abac
Figura 2. Papeles especiales (izquierda) obtenidos de las pastas de papel fabricadas por la
empresa CELESA (Tortosa, Tarragona) y sus principales materias primas (derecha).
8
1. Introduccin
Lignina
Hemicelulosas
Celulosa
9
1. Introduccin
1.2.1. Celulosa
La celulosa es el componente principal de las clulas vegetales, que
comprende aproximadamente del 10 al 20% del peso seco de las hojas, entre un
43 y un 47% de la madera de conferas, entre un 42 y un 44% de la madera de
frondosas y el 90% del peso de las fibras de algodn (Streitwieser y Heathcock
1983, Aitken et al. 1988). Estructuralmente, es un polmero lineal constituido
por unidades de -D-glucopiranosa unidas por enlaces glicosdicos (1o4), en
los que dos molculas de glucosa se unen con eliminacin de una molcula de
agua entre dos hidroxilos de los carbonos 1 y 4. La configuracin slo es
posible por la rotacin de la unidad de glucosa siguiente del eje C1-C4 del anillo
de piranosa, por eso la unidad de cadena de celulosa que se repite es la celobiosa
(disacrido), con una longitud de 1,03 nm (Fengel y Wegener 1984, Sjstrm
1993, Sjstrm y Westermark 1999). Los numerosos grupos hidroxilo favorecen
la formacin de enlaces de hidrgeno intra e inter-moleculares, formando cada
unidad de glucosa dos enlaces intramoleculares y uno intermolecular (Figura 4).
Los enlaces de hidrgeno intermoleculares se establecen con otras cadenas que
estn en el mismo plano, as como con cadenas en planos superiores e inferiores,
de este modo, las cadenas de celulosa se unen dando lugar a la formacin de
microfibrillas, y la unin de stas entre s a la fibra de celulosa, cuyos agregados
forman la pared celular (Lennholm y Henriksson 2007).
Unidad celobiosa
H
H
O
O
OH 1 4 OH 1
O HO O
HO
O O
O O
O
O
OH 1 4 HO O
HO OH 1 O
4
O
H H H
H
O
O
OH OH
O HO O HO
O O
O O
O
O O
OH HO OH
HO O O
H H
H
H
O
O
OH OH
O HO O HO
O O
O O
O
O HO O
OH
HO OH O
O
H H
10
1. Introduccin
1.2.2. Hemicelulosas
Las hemicelulosas comprenden aproximadamente del 25 al 30% del peso seco
de la madera de conferas, entre un 20 y 43% de la madera de frondosas, entre
un 12 y 18% de las fibras liberianas de lino y un 12% de las fibras de hojas de
sisal (Aitken et al. 1988, Fengel y Wegener 1984, Garca Hortal 2007). Actan
como matriz de soporte para las microfibrillas de celulosa y estructuralmente
son ms complejas que la celulosa.
11
1. Introduccin
H H OH H
H H O H O COOH H O
HO HO HO
HO OH HO OH HO OH
H H OH H OH
OH
H H H H H H
OH H OH H
COOH H
H H O OH O O
H HO H 3C O
HO H HO OH HO H
H OH H H OH
H
H OH H H H OH
O HO H OH
H H
O COOH
H O H O
HO H
H H
HO H HO H
H O H 2C OH H H
OH OH
H OH
H OH H OH
DESOXI-HEXOSAS
H
OH
H H O
H H O
HO
H
H H
H H
OH
OH
OH OH
OH OH
-L-Ramnosa -L-Fucosa
12
1. Introduccin
X- X - X - X
Glucuronoxilanos Frondosas, plantas no madereras
Ac Gl
7
G- M - M - M
Galactoglucomananos Conferas
Gal Ac
X - X - X- X - X5
Arabinoglucuronoxilanos Conferas, plantas no madereras
Gl A
2
1.2.3. Lignina
Despus de la celulosa, la lignina es el polmero ms abundante en el mundo
vegetal, representando entre un 25 y un 33% de la madera de conferas y entre
un 18 y un 34% de la madera de frondosas (Aitken et al. 1988). En el caso de las
plantas no madereras hay un menor porcentaje de lignina con respecto a las
especies madereras, con un 8-9% para fibras de hojas (abac y sisal), entre un 3-
13% para fibras liberianas (lino, camo, yute y kenaf), entre un 12 y un 21%
para pajas (paja de arroz, paja de trigo) y entre un 19 y un 22% para caas
13
1. Introduccin
14
1. Introduccin
OH OH OH O
J
O
OH OH OH OH
I II III IV
OH
O OH H O
OH OH OH
V VI VII
15
1. Introduccin
OH
J OH
OH OH OH OH
Peroxidasa
Lacasa
- (e- + H+)
OMe
OMe
OMe OMe
OH OMe OMe
O
O O O O
a
Lignina Lignina
(MeO) (MeO)
Lignina Lignina
(MeO)
HO HO HO (MeO)
HO O HO
OMe OMe
R
O O
OMe
Acoplamiento OMe
Oxidacin radicalar ROH
(MeO) OMe (MeO) OMe (MeO) OMe
(MeO) OMe
OH O O OH
16
1. Introduccin
6
MeO 1
HO MeO 5
6 2
J 5 1 OH
J 4 3
D E E
4 2
HO 3
O HO D O
OMe
OMe OMe
1 1 2 1
6 2 6 2 1 3 6 2
5 3 5 3 6 4 5 3
4 4 5 4
MeO OMe OMe O OMe
OMe
O O OMe O
OMe
1
6 2
O
5 3 3
HO 2 4
4
OMe J
1 5
O
HO
E E 6
J HO D OMe J D
OMe
D O 2
1 3
E E
6 4
5
1 1
O MeO D J
6 2 6 2 6
5 1 O
5 3 5 3
4 4 OMe 4 2
MeO OMe MeO OMe 3
O
O O
OMe
1 1
2 6 6 2
3 5 5 3
4 4 OMe
MeO OMe
O 1''
6'' 2''
3
4
O O 5'' 3''
2
4''
5 MeO OMe
Dcc Ecc MeO 1
6 D
O O
OH J E D E
6 1
5 J
MeO 2 OH
HO
1
4 3 6 2
5 3
OMe 4
O MeO OMe
O
17
1. Introduccin
A B
(a) (b)
Figura 9. Modelos del polmero de lignina en maderas: (a) confera (Picea) (Brunow 2001) y
(b) frondosa (lamo) (Boerjan et al. 2003).
18
1. Introduccin
OMe OH
OH O OH
HO O
OMe
O OH
O O MeO OMe
OMe O O O
O
OH OMe O
HO HO
OMe OH OH
O
O OMe
HO
OMe
MeO OMe
O
O O O
O O
O MeO OMe
OMe O
HO
OH
O
O OMe
O
O MeO OMe
O
O
MeO OMe OMe
O HO HO
OH
OMe O
HO HO OMe OH
O O
O OH
O
O MeO OMe
O OMe
HO
MeO OMe O
O O
O O
OMe OMe
HO OH
MeO
O
OMe OH O OMe
O
OH
HO O
HO
MeO OMe OMe
O OMe
HO
O MeO O
O
OMe O OH
OH O
MeO MeO
O OMe O OMe
O
HO O HO OH
OMe OMe
O O
O OH O OH
O O
MeO MeO
O OMe O OMe
HO OH
HO O
O O OH
OMe
OMe
MeO O OMe
O
MeO O
O O OH OH
O HO
OMe MeO
OH OMe MeO HO
O O OMe
MeO O O O
O OH
O OH OH
MeO MeO
HO OMe HO OMe
(a) (b)
Figura 10. Modelos del polmero de lignina en plantas no madereras: (a) kenaf y (b) abac
(del Ro et al. 2008a).
19
1. Introduccin
20
1. Introduccin
OH
a b
O O
H OH
c d
COOH
HO
e fF
O
g
CO-O-CH2
CO-O-CH
CO-O-CH2
CH2OH
O O
OH O
O HO
OH j
i
21
1. Introduccin
Por otro lado, los extrables polares engloban diferentes compuestos fenlicos
libres de bajo peso molecular (Figura 12), los cuales incluyen precursores de la
lignina (cidos p-hidroxicinmicos y aldehdos p-hidroxicinamlicos), cidos
bencenocarboxlicos relacionados (cido p-hidroxibenzoico, vainllico y
sirngico), aldehdos y cetonas aromticas (p-hidroxibenzaldehdo, vainillina,
siringaldehdo y propioguayacona), e incluyen taninos hidrolizables (steres del
cido glico y sus dmeros), flavonoides (estructuras derivadas del anillo de
flavona, 2-fenilbenzopirona) y taninos no hidrolizables (varias unidades de
flavonoides condensadas). Adems de incrementar el consumo de reactivos
durante la coccin, estos compuestos pueden dificultar las reacciones de
pasteado impidiendo la difusin de los reactivos en la materia prima, y los
taninos, cuando estn presentes en cantidades importantes, forman complejos
coloreados con cationes metlicos afectando el color de las pastas de papel y su
blanqueabilidad (Garca Hortal 2007).
HO O
HO O O H O
O O O O O
OH OH OH OH
a b c d
HO O
HO OH
OH
O
e f
22
1. Introduccin
23
1. Introduccin
Procesos mecnicos
El pasteado mecnico tiene como objeto la separacin fsica de las fibras,
realizndose el desfibrado por fragmentacin mecnica, utilizando molinos y
refinadores de discos. La fabricacin de pastas mecnicas ofrece la ventaja de
dar como resultado rendimientos elevados (hasta un 98% del material inicial),
obtenindose pastas ventajosas para algunos tipos de papel por su rigidez,
volumen y opacidad (Garca Hortal 2007). Sin embargo, como en este proceso la
lignina slo se ablanda (no se disuelve), el alto contenido en lignina va en
detrimento de la calidad del papel ya que las fibras muy lignificadas son rgidas,
poco flexibles, no estn bien unidas entre s, proporcionando papeles con bajas
caractersticas de resistencia y muy sensibles al envejecimiento ptico.
Procesos qumicos
En el pasteado o coccin qumica, la deslignificacin se lleva a cabo con la
ayuda de agentes qumicos cidos o bsicos, en digestores o reactores a altas
temperaturas y presiones. La pasta se produce con disolucin de la lignina que
se encuentra entre las fibras del material lignocelulsico y los productos de
degradacin se disuelven en la leja de la coccin. En el pasteado qumico, se
eliminan muchos de los componentes no fibrosos de la materia prima y los
rendimientos son normalmente del 35 al 65%, sin embargo, la pasta se blanquea
mejor y el producto es ms resistente y de mejor calidad que en el caso de los
procesos mecnicos (Sjstrm 1993).
24
1. Introduccin
25
1. Introduccin
60%. En estos procesos tambin se degradan los hidratos de carbono por rotura
de los enlaces glicosdicos, lo que provoca una disminucin del grado de
polimerizacin todava mayor que en los procesos kraft siendo la pasta
resultante menos resistente, pero por lo contrario estas pastas son ms fciles de
blanquear. El mtodo al sulfito ha sido relegado en parte por el proceso kraft
(Bryce 1990).
26
1. Introduccin
27
1. Introduccin
La problemtica del pitch es muy compleja porque vara con la materia prima
as como con el proceso empleado para la fabricacin de pasta y papel. En el
caso de las pastas mecnicas, los depsitos de pitch muestran una composicin
similar a los extractos lipoflicos de la materia prima. En el caso de las pastas
alcalinas, slo algunos de los compuestos extrables presentes en la materia
prima sobreviven al proceso de coccin. En condiciones alcalinas los steres de
glicerol se saponifican y los cidos grasos y resnicos se disuelven. Los steres
de esteroles, los esteroles libres y las ceras, se saponifican ms lentamente que
los steres de glicerol, no forman jabones solubles como en el caso de los cidos
libres, por lo que tienen tendencia a depositarse (Gutirrez et al. 2001).
28
1. Introduccin
Figura 14. Imagen de una gota de resina en el rbol (izquierda) y de un depsito de pitch
en una pasta kraft TCF (cedidas por Mara Jess Ortega y Javier Romero, respectivamente).
29
1. Introduccin
30
1. Introduccin
Figura 15. Enzimas de inters en la industria de la pasta y papel: (a) xilanasa, (b) lacasa y (c)
peroxidasa verstil.
31
1. Introduccin
Alcohol Hlice B
veratrlico
(VA) VA
Trp164 Glu36
VP VA
[Fe3+]
Glu40 ROOH Mn3+ C-IIB
[Fe3+ Trp ]
Mn2+ ABTS+
agua Mn2+
ROH
C-IA C-IIA
[Fe4+=O P] [Fe4+=O]
Mn2+ Mn3+
VA
VA
C-IB
ABTS [Fe4+=O Trp ]
(fenoles)
(a) (b)
Figura 16. Vista axial de la regin del hemo de la VP que muestra los tres sitios de la
oxidacin: Mn2+ (que incluye tres residuos acdicos de amino-cidos), ABTS (en el lmite del
grupo hemo) y VA (a travs de Trp164) (a) y ciclo cataltico propuesto para la VP (b) (Ruiz-
Dueas y Martnez 2010, Prez-Boada et al. 2005).
32
1. Introduccin
H2O
H2O2
Compuesto 1
(enzima activa)
POMred
H2O Mn2+
Ligninaox
Mn3+
POMox Lignina
Mn3+
POMred POMox
Lignina
Mn2+
Ligninaox
Compuesto 2
(a) (b)
33
1. Introduccin
Por otro lado, las lacasas constituyen un grupo de enzimas oxidativas que
ha sido objeto de gran inters en el desarrollo de tecnologas respetuosas con el
medio ambiente (Mayer y Staples 2002). Como se ha comentado anteriormente,
la accin directa de las lacasas en principio est restringida a las unidades
fenlicas, pero en presencia de mediadores redox (Bourbonnais y Paice 1990,
Call 1994) amplan la accin de la lacasa a sustratos no fenlicos, lo que
aumenta su potencial en la degradacin de lignina y de otros compuestos
aromticos. Recientemente, se ha mostrado la gran eficacia del sistema lacasa-
mediador en la eliminacin de extrables lipoflicos de pastas de conferas,
frondosas as como de fibras no madereras (Gutirrez et al. 2006a, 2006c,
2006b, 2007, 2009, Molina et al. 2008).
(a) (b)
34
1. Introduccin
35
2
OBJETIVOS
37
2. Objetivos
39
3
MATERIAL Y MTODOS
3.1. MATERIALES
En esta Tesis se ha estudiado la composicin qumica de fibras de diversos
cultivos agrcolas utilizadas para la fabricacin de pastas de celulosa de alta
calidad y el comportamiento de sus componentes a lo largo del proceso de
fabricacin de dichas pastas. Entre los materiales estudiados se encuentran fibras
del tallo de varias plantas anuales, tales como lino, kenaf, camo y yute; y
fibras procedentes de hojas de sisal, abac y curau. Tambin se seleccionaron
pastas crudas y pastas blanqueadas (TCF y ECF). Tanto las muestras
correspondientes a las materias primas como sus pastas fueron suministradas por
la empresa Celulosa de Levante S.A., CELESA (Tortosa, Tarragona). Por otro
lado, tambin se estudiaron otras fibras de potencial aplicacin en el sector
procedentes de tallos de caa comn, as como fibras de residuos de poda de
rboles de tagasaste, que fueron suministradas por la Universidad de Huelva.
Finalmente, para la realizacin de los diversos tratamientos biotecnolgicos se
utilizaron pastas de eucalipto y de lino, suministradas por las empresas ENCE
(Pontevedra) y CELESA, respectivamente.
Lino
El lino (Linum usitatissimum) (Figura 19) es una planta herbcea
dicotilednea de la familia de las Linceas, originaria de Asia que se cultiva por
el aceite de su semilla y por las fibras de sus tallos. Se cultiva principalmente en
regiones fras y templadas como Europa, Asia, Australia, Argentina y Brasil.
Figura 19. Planta de lino (izquierda) y morfologa de sus fibras elementales (derecha) (Garca
Hortal 2007).
41
3. Material y Mtodos
Camo
Figura 20. Planta de camo (izquierda) y morfologa de sus fibras elementales (derecha)
(cedida por Garca Hortal).
42
3. Material y Mtodos
Kenaf
El kenaf (Hibiscus cannabinus) (Figura 21) es una planta herbcea
dicotilednea de la familia de las Malvceas, probablemente originaria de
frica. Se cultiva en regiones tropicales y subtropicales de la India, Sudeste de
Asia y Amrica Central. Es una planta de crecimiento rpido, que se est
desarrollando como fuente de fibras para la fabricacin de papel.
Figura 21. Planta de kenaf (izquierda) y morfologa de sus fibras elementales (derecha)
(Garca Hortal 2007).
43
3. Material y Mtodos
Yute
El yute (Corchorus capsularis) (Figura 22) es una planta herbcea
dicotilednea de la familia de las Tiliceas, que crece principalmente en climas
hmedos y clidos de Paquistn, India, Bangladesh, Brasil y otros pases
tropicales. Sus haces fibrosos, que tienen una longitud de 20 a 50 cm y
contienen de 10 a 30 fibras elementales, se usan principalmente para la
produccin de sacos, tapices, cuerdas, textiles y materiales para embalaje.
Las plantas de yute presentan tallos que pueden alcanzar alturas de 2,5-3 m y
10-20 mm de dimetro. La fibra elemental tiene una longitud variable de 1,5 a 5
mm, el menos elevado de las otras fibras liberianas mencionadas, y un ancho de
10 a 25 m. Los tallos son enriados en estanques de agua, donde se liberan los
haces de fibras liberianas de la corteza y la porcin leosa de la planta (Garca
Hortal 2007, Han 1998).
Figura 22. Planta de yute (izquierda) y morfologa de sus fibras elementales (derecha)
(Garca Hortal 2007).
44
3. Material y Mtodos
Sisal
El sisal (Agave sisalana) (Figura 23) es una planta robusta de climas
tropicales originaria de Amrica Central y Mxico, cultivada actualmente en
Brasil, Venezuela, Tanzania, Kenia, Mozambique, Angola, Madagascar y otras
zonas tropicales. Es una planta monocotilednea de la familia de las Agavceas
y sus fibras, denominadas fibras duras, ms rgidas y bastas que las fibras
liberianas, se han utilizado durante mucho tiempo en las industrias textil y de
cordelera.
Figura 23. Plantas de sisal (izquierda) y morfologa de sus fibras elementales (derecha)
(Garca Hortal 2007).
45
3. Material y Mtodos
Abac
El abac (Musa textilis) (Figura 24) es una planta perenne nativa de Filipinas
y que se cultiva actualmente tambin en Indonesia y Amrica tropical. Es una
planta monocotilednea que pertenece a la familia de las Musceas y sus fibras
tambin estn clasificadas como fibras duras.
Los papeles producidos por las fibras de abac son altamente porosos, por lo
que son ideales para filtros y envolturas de embutidos. Se considera una
excelente materia prima para papeles de alta calidad, como papel moneda,
paales, servilletas, papel tis, accesorios para hospitales, etc. El abac de
primera calidad tambin se emplea mezclado con algodn o pasta de madera en
la fabricacin de papeles superfinos, papel para imprimir de bajo gramaje, de
registro, moneda y seguridad y, sobre todo, papel para filtro poroso de uso en
laboratorio o industrial.
Figura 24. Planta de abac (izquierda) y morfologa de sus fibras elementales (derecha)
(cedida por Garca Hortal).
46
3. Material y Mtodos
Curau
El curau (Anans erectifolius) es una planta herbcea de fruto no comestible
(Figura 25a) perteneciente a la familia de las Bromeliceas, de origen americano
y hbitat tropical, muy comn en el Amazonas. Tiene un tallo tan corto que
parece carecer de l y con hojas rgidas. Normalmente produce entre 20 y 24
hojas, proporcionando aproximadamente 2 kg de fibra. En la ltima dcada, ha
ganado reconocimiento comercial como material para composites en la industria
del automvil (Leao et al. 1998, Silva et al. 2001). La fibra del curau tambin
se ha propuesto como una materia prima alternativa para la produccin de pastas
qumicas en Brasil.
Caa comn
La caa comn (Arundo donax) es una planta monocotilednea perenne,
perteneciente a la familia de las Poceas que parece ser originaria de Asia y que
ha colonizado el rea del Mediterrneo, en pases como Portugal y Espaa. Es
considerada una de las mayores gramneas, con una estructura tubular
segmentada semejante al bamb (Figura 25b), con alturas entre 2 y 8 m (Seca et
al., 2000). Debido a la fcil adaptabilidad de esta gramnea a diferentes
condiciones ecolgicas, a la elevada productividad de biomasa y capacidad de
cultura intensiva, es una de las especies no madereras ms atractivas como
fuente alternativa de fibras para la industria de pasta de papel (Shatalov y
Pereira 2002).
Tagasaste
El tagasaste (Chamaecytisus proliferus spp. palmensis) es un arbusto robusto
de crecimiento rpido perteneciente a la familia de las Fabceas. Es nativo de las
Islas Canarias y se cultiva en Australia, Nueva Zelanda y otros pases. Debido a
su alto contenido proteico, se usa como alimento para el ganado y tambin como
cultivo fijador del nitrgeno para mejorar la fertilidad del suelo. Con el fin de
fomentar la formacin de tallos mltiples, el arbusto debe ser podado con
regularidad, lo que conduce a una alta acumulacin de residuos de poda. Estos
residuos se han evaluado recientemente como materia prima alternativa para la
produccin de pasta de celulosa (Daz et al. 2004, Lpez et al. 2004, Jimnez et
al. 2006, 2007, Garca et al. 2008). En la Figura 25c se muestra una fotografa
de un arbusto de tagasaste.
47
3. Material y Mtodos
a c
Figura 25. Diversas plantas estudiadas como materias primas potenciales para la produccin
de pasta de celulosa: (a) curau, (b) caa comn y (c) tagasaste.
48
3. Material y Mtodos
Lipoxigenasas
La lipoxigenasa utilizada se obtuvo del hongo ascomiceto Gaeumannomyces
graminis y fue suministrada por la empresa Novozymes (Bagsvaerd,
Dinamarca). La unidad de actividad de esta enzima se define como la cantidad
de enzima que da lugar a un aumento en la Absorbancia a 234 nm, de 0,001 por
minuto a pH 7 y 30C, cuando se usa el cido linoleico como sustrato (volumen
de reaccin = 1,0 mL y camino ptico = 1 cm). Los tratamientos enzimticos
con lipoxigenasa incluyeron reacciones con varios lpidos modelo, como alcanos
(nonacosano), alcoholes (octacosanol), cidos grasos (cido linoleico), esteroles
libres (sitosterol) y steres de esteroles (colesteril linoleato), representativos de
los extrables lipoflicos de las pastas estudiadas. El sitosterol fue suministrado
por Calbiochem y los dems compuestos por Sigma-Aldrich.
Peroxidasas
Se utiliz la peroxidasa verstil del hongo basidiomiceto Pleurotus eryngii
(VP), producida en el Centro de Investigaciones Biolgicas (CIB, CSIC,
Madrid) como se describe a continuacin.
49
3. Material y Mtodos
Una vez lavados los cuerpos de inclusin con una solucin de Tris-HCl pH
8,0 20 mM, EDTA pH 8,0 1 mM y DTT 5 mM, se resuspendieron en el mnimo
volumen posible de la misma solucin de lavado, se homogenizaron y
centrifugaron. Se resuspendieron de nuevo los cuerpos de inclusin en el
mnimo volumen de solucin desnaturalizante (solucin de Tris-HCl pH 8,0 50
mM, EDTA pH 8,0 1 mM, DTT 5 mM y urea 8 M), se incubaron a temperatura
ambiente durante 15 min y se centrifugaron para eliminar los restos no disueltos.
El sobrenadante, con la protena completamente desplegada, se diluy 5 veces
con una solucin de Tris-HCl pH 8,0 50 mM, EDTA pH 8,0 1 mM y DTT 1
mM, para llegar a una concentracin final de urea de 1,6 M y de 1-2 mg/mL de
protena. Esta solucin se aadi a la mezcla de replegado (CaCl2 5 mM,
glutatin oxidado 0,5 mM, Tris-HCl pH 9,5 50 mM y hemina 20 M) en una
proporcin 1:10 para obtener una concentracin final de urea de 0,16 M y se
incub en oscuridad a temperatura ambiente durante 16 horas.
Polioxometalatos
El POM utilizado fue [SiW11MnIII(H2O)O39]5- denominado SiW11MnIII en
forma simplificada. La solucin acuosa del POM se prepar a partir de
-K8[SiW11O39]13H2O, KMnO4 y Mn(CH3COO)24H2O. Se disolvieron 9,6 g
de K8[SiW11O39]13H2O en 13 mL de agua, a 95C. En vasos separados se
disolvieron, a temperatura ambiente, 0,095 g de KMnO4 en 10 mL de HCl
0,6mM y 10 mL de agua y 0,6 g de Mn(CH3COO)24H2O en 10 mL de agua. Al
50
3. Material y Mtodos
51
3. Material y Mtodos
Esteroles
Hidrocarburos
esteroidales
steres de esteroles
Escualeno Triglicridos
cidos grasos
10 20 30 min
Fase aminopropilo
Acondicionamiento
A B C D
Escualeno
steres de
Hidrocarb. esteroles
esteroidales
Ceras
Triglicridos Esteroles
10 20 30 min
C16
C18:2
cidos grasos
10 20 30 min
C18:1
C26
10 20 30 min C18
C22C24 C28C
C20 30
10 20 30 min
Figura 26. Esquema del fraccionamiento de un extracto lipdico por SPE (Gutirrez et al.
2004).
52
3. Material y Mtodos
53
3. Material y Mtodos
54
3. Material y Mtodos
55
3. Material y Mtodos
56
3. Material y Mtodos
lignina (Lu y Ralph 1997a, 1997b, 1998). Una de las ventajas de este mtodo es
que deja intacto el carbono de la cadena lateral de la lignina, por lo que lo hace
muy adecuado para estudiar la presencia de unidades aciladas (con acetatos, p-
cumaratos o p-hidroxibenzoatos) en el carbono . Sin embargo, el mtodo de
DFRC usa reactivos acetilantes que interfieren en el anlisis de los grupos
acetatos nativos en la lignina, pero con las modificaciones apropiadas, mediante
la sustitucin de la acetilacin por la propionilacin (DFRC), es posible obtener
una informacin significativa sobre la presencia de unidades acetiladas en la
lignina nativa (Ralph y Lu 1998). En la Figura 27a se puede observar la rotura
selectiva de los enlaces ter en el mtodo DFRC y en la Figura 27b la
modificacin de este mtodo (DFRC) para el anlisis de ligninas naturalmente
acetiladas.
57
3. Material y Mtodos
AcBr Zn
Ac2O/Py
CH3O OCH3 CH3O OCH3 CH3O OCH3
O O OAc
RO RO OR
HO Br
O O
AcBr Zn
Ac2O/Py
CH3O OCH3 CH3O OCH3 CH3O OCH3
O O OAc
(b)
HO PropO OProp
HO Br
O O
PropBr Zn
Prop2O/Py
CH3O OCH3 CH3O OCH3 CH3O OCH3
O O OProp
HO Br
O O
PropBr Zn
Prop2O/Py
CH3O OCH3 CH3O OCH3 CH3O OCH3
O O OProp
Figura 27. Rotura selectiva de los enlaces ter: (a) Mtodo DFRC (Lu y Ralph 1997a) y (b)
Mtodo DFRC modificado (DFRC) para el anlisis de ligninas naturalmente acetiladas
(Ralph y Lu 1998).
58
3. Material y Mtodos
0
0
Regin
aliftica
50
50
Regin aliftica
C (ppm)
oxigenada
100
100
Regin
aromtica
150
150
ppm (t1
10
10.0 5
5.0 H (ppm) 0
0.0
ppm (t2)
Figura 28. Espectro HSQC de la lignina aislada de sisal donde se pueden observan sus tres
regiones caractersticas.
59
3. Material y Mtodos
-OMe J
J-OAc J-OH
D
E (J-Oac)
D D
E (J-OH)
R O
HO MeO
O MeO J 6
6 5 1
J 1
5
HO E 4 2
HO E 4 2 D 3
D 3 O
O
OMe
OMe 6
1
2
1
6 2 3
5
5 3 4
4 MeO OMe
MeO OMe
O
O
OMe
OMe
O O 1
3
2 4 6 2
3
4 3
5
O 1
6
5 2 4
J D OMe MeO
5 1 MeO OMe
6
D O
O
E E D
J E E
MeO D J
6
O HO J OH
5 1 1
6 2
4 2
3 5 3
O MeO
4
OMe
OMe O
Figura 29. Regin aliftica oxigenada del espectro HSQC de la lignina aislada de sisal y sus
correspondientes estructuras.
60
3. Material y Mtodos
S2,6 S2,6
S2,6
G2 2
G5
G6 6'
O O OH
HO
D D
D
S S S
1 1
1 6 2 6 2
6 2
5 3 5 3
5 3 4 4
4 MeO OMe MeO OMe
MeO OMe
OH O
O
OMe
HO O 1
6 2
3
D 4
5 3
2 4
MeO
5 1 MeO OMe
G
1 6
6 2 D O
3
O
5
4 J E D E
OMe
HO J OH
O 6
1
2
5 3
4
MeO
OMe
O
Figura 30. Regin aromtica del espectro HSQC de la lignina aislada de sisal y sus
correspondientes estructuras.
61
3. Material y Mtodos
62
3. Material y Mtodos
63
3. Material y Mtodos
64
3. Material y Mtodos
Siendo,
65
3. Material y Mtodos
Una vez tratadas las pastas, se extrajeron con acetona en un extractor de tipo
Soxhlet durante 6 horas. A continuacin se evapor el disolvente a sequedad en
un rotavapor y los extractos lipoflicos obtenidos se redisolvieron en CHCl3 para
su posterior anlisis mediante GC y GC/MS, en las condiciones descritas
anteriormente para el anlisis de los compuestos lipoflicos. Se realizaron
anlisis posteriores de las propiedades de las pastas (blancura ISO, ndice kappa,
viscosidad intrnseca y cidos hexenurnicos).
66
3. Material y Mtodos
67
3. Material y Mtodos
Una vez conocidas las condiciones ptimas para la reoxidacin del POM, se
procedi a la realizacin de los diferentes ensayos de deslignificacin. Para cada
uno de los ensayos, se dej la pasta (8 g) en agua destilada (600 mL) durante
una noche en agitacin constante. Se filtr, se pas al reactor y se adicion 67
mL de tampn acetato de sodio 0,2 M pH 4,5 (concentracin final de 0,1 M),
disolucin de POM concentrado (entre 12,5 a 13,5 mL para que la concentracin
final de POM en la mezcla fuera de aproximadamente 3 mM) y agua destilada
hasta 134 mL de volumen total (considerando todava la contribucin del agua
retenida en la pasta filtrada), para obtener una consistencia final del 6%. Se
presuriz el reactor con pO2=5 bar, se encendi la agitacin mecnica y se llev
hasta la temperatura de 110C. Una vez acabado el ensayo, se filtr la pasta y se
lav con agua destilada (POM 1-). En los casos en que los lquidos de filtrado
conteniendo el POM fueron reoxidados (-VP-), se adicion a estos la enzima y
el perxido de hidrgeno en las cantidades necesarias (obtenidas a travs de los
ensayos de optimizacin descritos anteriormente), manteniendo agitacin
constante y a temperatura ambiente (para no desnaturalizar la enzima), durante
aproximadamente 20 min (aunque a los seis minutos la reoxidacin ya se
estimaba como completa). El filtrado conteniendo el POM reoxidado se aadi
nuevamente a la pasta filtrada reiniciando una nueva etapa de deslignificacin
(-POM 2-).
68
3. Material y Mtodos
(a) (b)
69
3. Material y Mtodos
pu f
IK >1 0,013(25 t )@
w
(b a) N
p
0,1
Con:
IK = ndice kappa.
70
3. Material y Mtodos
rel = htn
Con:
A partir de la tabla que se encuentra en anexo (Anexo 4), se lee el valor del
producto []C, que corresponde al valor de la viscosidad relativa obtenido:
rel = []C
Con:
71
3. Material y Mtodos
72
3. Material y Mtodos
73
4
REFERENCIAS
75
4. Referencias
76
4. Referencias
77
4. Referencias
78
4. Referencias
79
4. Referencias
80
4. Referencias
81
4. Referencias
82
4. Referencias
83
4. Referencias
84
4. Referencias
85
4. Referencias
86
4. Referencias
87
4. Referencias
Zhang, L. M., Gellerstedt, G., Ralph, J. y Lu, F. C. (2006) NMR studies on the
occurrence of spirodienone structures in lignins. Journal of Wood Chemistry and
Technology, 26, 65-79.
Zhang, X., Nguyen, D., Paice, M. G., Tsang, A. y Renaud, S. (2007) Degradation
of wood extractives in thermo-mechanical pulp by soybean lipoxygenase.
Enzyme and Microbial Technology, 40, 866-873.
88
4. Referencias
89
5
RESULTADOS Y DISCUSIN
91
5. Resultados y discusin
Publicacin I:
Marques G., Rencoret J., Gutirrez A., del Ro J.C. (2010) Evaluation of the
chemical composition of different non-woody plant fibers used for pulp and paper
manufacturing. The Open Agriculture Journal (in press).
92
5. Resultados y discusin
Instituto de Recursos Naturales y Agrobiologa de Sevilla, CSIC, P.O. Box 1052, 41080-
Seville, Spain
Abstract
The chemical composition of several non-woody plant fibers (bast fibers from
flax, hemp, kenaf, jute; leaf fibers from sisal, abaca and curaua; and giant reed),
which are used as raw materials for pulp and papermaking, has been evaluated.
Particular attention was paid to the composition of the lipophilic compounds and
the structure of the lignin polymer since they are important components of the
fiber that strongly influence the pulping and bleaching performances.
Keywords: non-woody fibers; flax, hemp, kenaf, jute, sisal, abaca, giant reed,
paper pulp; lipophilic extractives; lignin
1. Introduction
An alternative to woody raw materials for pulp and paper production in
developing countries is the use of non-woody fibers from field crops and
agricultural residues. In developed countries, non-woody fibers are mainly used
for the production of specialty papers, i.e., tea bags, filter papers, bank notes,
etc. On the other hand, there is a growing need within Europe to consider
alternative agricultural strategies that move an agricultural industry purely
focused on food production to one that also supplies the needs of other industrial
sectors, such as paper and textiles. Non-wood fibers, therefore, could become
important raw materials in this transformation [1-3]. The main sources of non-
woody raw materials are agricultural residues from monocotyledons, including
cereal straw and bagasse, or plants grown specifically for the fiber, such as
bamboo, reeds, and some other grass plants such as flax, hemp, kenaf, jute, sisal,
or abaca. Non-woody plants offer several advantages including short growth
cycles, moderate irrigation requirements and low lignin content, which in
principle would result in reduced energy and chemicals consumption during
pulping [4].
Plant fibers are constituted by three structural polymers (the polysaccharides
cellulose, and hemicelluloses and the aromatic polymer lignin) as well as by
some minor non-structural components (i.e. proteins, extractives, minerals).
Pulping and bleaching performances are highly dependent on the relative
content, structure and reactivity of the plant components. In particular, the lignin
content and its composition in terms of p-hydroxyphenyl (H), guaiacyl (G) and
syringyl (S) moieties and the different inter-unit linkages are important factors
93
5. Resultados y discusin
in pulp production affecting the delignification rate. It has been shown that
higher S/G ratios in woods implied higher delignification rates, less alkali
consumption and therefore higher pulp yield [5]. On the other hand, among the
non-structural components, lipophilic extractives present special relevance due
to their high impact in paper pulp manufacturing [6]. Lipophilic extractives
include different classes of compounds (i.e. alkanes, fatty alcohols, fatty acids,
free and conjugated sterols, terpenoids, triglycerides and waxes), which have
different behavior during pulping and bleaching [6-8]. These lipophilic
compounds, even when present in low amounts in the raw material, may play an
important role during the industrial wood processing for pulp and paper
production since they are at the origin of the so-called pitch deposits. Pitch
deposition is a serious problem in the pulp and paper industry being responsible
for reduced production levels, higher equipment maintenance costs, higher
operating costs, and an increased incidence of defects in the finished products,
which reduces quality and benefits [6].
In order to maximize the exploitation of non-woody plant fibers for paper
pulp production, a more complete understanding of its chemistry is required.
Most studies have been devoted to the chemical characterization of woody
materials, while studies on non-woody fibers have been comparatively scarce. In
this context, the main objective of this work is to revise and evaluate the
chemical composition of different non-woody plant fibers used for pulp and
papermaking, that will help improving the industrial processes in which they are
used as raw materials.
2. Analytical methodologies
2.1. Samples
The samples selected for this study were bast fibers from flax (Linum
usitatissimum), hemp (Cannabis sativa), kenaf (Hibiscus cannabinus) and jute
(Corchorus capsularis); leaf fibers from sisal (Agave sisalana), abaca (Musa
textilis) and curaua (Ananas erectifolius); as well as giant reed (Arundo donax).
94
5. Resultados y discusin
95
5. Resultados y discusin
length), similar to the short fibers of hardwoods, while others, and particularly
flax and hemp bast fibers, present remarkably high lengths (up to 28000 m
fiber length).
Table 1. Morphological characteristics (length and width) of the selected fibers [19].
Fiber Source Length (m) Width (m) L/W ratio
Bast fibers
flax 28000 21 1350:1
hemp 20000 22 1000:1
kenaf 2740 20 135:1
jute 2000 20 100:1
Leaf fibers
sisal 3030 17 180:1
abaca 6000 20 300:1
curaua n.a. n.a. n.a.
Reeds
giant reed 1180 15 78:1
Among the studied fibers, flax and hemp pulps have traditionally been used as
the primary furnish for cigarette paper (burning tube), where strength, opacity
and control of air permeability are required. Banknote paper often incorporates
flax or hemp to enhance general strength characteristics. Jute pulp is used for
high porosity papers. Its fiber length plus low diameter makes it very suitable for
finishing paper purposes. Sisal and abaca pulps have an unusually high tearing
resistance and high porosity and are well suited for the production of papers
where high strength and high porosity are required.
96
5. Resultados y discusin
Table 2. Composition of the main constituents of the selected fibers (% of dry matter) [20-
22].
Ash Acetone Water-solubles Klason Acid-soluble Holocellulose
extractives lignin lignin
Bast fibers
flax 1.5 0.7 1.3 2.9 1.6 92.0
hemp 2.0 0.5 1.2 4.6 1.5 90.3
kenaf 1.8 1.0 1.1 11.4 3.0 81.9
jute 2.4 0.5 0.4 13.3 2.8 81.6
Leaf fibers
sisal 1.0 0.7 2.3 5.9 3.0 85.0
abaca 0.9 0.5 1.7 7.7 1.4 85.6
curaua 1.3 5.3 5.1 4.9 1.6 92.5
Reeds
giant reed 4.2 1.6 8.5 24.7 n.d. 49.8
97
5. Resultados y discusin
Bast fibers
flax 0.4 0.9 1.1 8.8 3.5 85.3
hemp 0.4 0.6 1.0 9.9 1.6 86.4
kenaf 0.5 2.1 10.5 4.9 0.5 81.5
jute 0.5 1.5 7.9 4.2 0.5 85.4
Leaf fibers
sisal 0.3 1.9 12.0 3.6 0.6 81.7
abaca 0.3 1.6 7.5 3.5 0.3 86.9
curaua 0.0 2.7 8.0 3.5 0.2 85.6
Reeds
giant reed 0.0 3.4 39.2 0.3 0.8 56.3
98
5. Resultados y discusin
kenaf and jute are fatty acids (28 and 35% respectively), followed by ester
waxes (26 and 27% respectively). Among the selected leaf fibers, free sterols
and fatty acids predominate in both sisal (20 and 24%, respectively) and abaca
(45 and 19%, respectively), while in curaua fibers, fatty acids and ester waxes
predominate (38 and 34%, respectively), followed by free sterols (10%). Finally,
in giant reed, the predominant lipophilic compounds are fatty acids (40%),
followed by free sterols (19%) and ester waxes (15%).
Generally, these fibers are pulped by an alkaline process, usually
soda/anthraquinone pulping. Therefore, we discuss the behavior and fate of the
different fiber components during alkaline cooking. In this context, the lipids
present in these fibers can be classified, in general terms, into two principal
groups, namely fatty acids (including - and
-hydroxyfatty acids) and neutral
components, including wax esters, long-chain n-fatty alcohols, alkanes, and
steroids and triterpenoids. The different lipids classes have different behavior
during cooking and bleaching [7, 8]. The wax esters, which are abundant
lipophilic compounds in some of these fibers (i.e. flax and curaua), are
hydrolyzed during alkaline cooking and the fatty acids dissolved. At sufficiently
high pH (as in alkaline pulping), the acids dissociate and form fatty acid soaps
and can thus dissolve in water to quite a high extent. By contrast, alkanes, fatty
alcohols, sterols and triterpenols, steroid hydrocarbons and ketones, and steryl
glycosides do not form soluble soaps under the alkaline pulping conditions and
therefore survive cooking. These compounds have a very low solubility in water
and are difficult to remove, and therefore can be at the origin of pitch deposition.
The low amounts of these neutral compounds in most of the fibers, and
particularly the low abundances of free and conjugated sterols, which have a
high propensity to form pitch deposits [28-30] would point to a low pitch
deposition tendency of the lipophilics from these fibers. On the other hand, fatty
acid soaps are effective solubilizing agents facilitating the removal from pulp of
these sparingly soluble neutral substances. Therefore, the ratio of saponifiables-
to-unsaponifiables has been suggested to be a better index for predicting pitch
problems than the total amount of lipids (Back and Allen, 2000). In fact, the
higher abundances of unsaponifiable compounds (neutrals) with respect to the
saponifiable ones is the main cause for pitch problems during pulping of some
woods, such as aspen or eucalypt [28-31]. Fatty alcohols, alkanes and sterols are
among the compounds responsible for pitch deposits formed during pulping of
nonwoody plants [8, 32]. In most of the fibers, as in flax, hemp, kenaf or jute
fibers, the content of free fatty acids (including - and
-hydroxyfatty acids) is
high, and therefore the fatty acid soaps formed during alkaline pulping may
possess sufficient micellar-forming properties to carry the less polar compounds
into solution. However, in other fibers, such as sisal and particularly abaca, the
fatty acids amounts up to only 20% of total lipophilic compounds, and therefore
they would be more prone to produce pitch deposition.
99
5. Resultados y discusin
OH
A B
O O
H OH
C D
O
HO
OH
E
O
OH
F OH
O
G H
HO
CH2OH
O
O
OH O
HO
I J K
OH
O
L
O O
O
O-CH2
HO-O-CH
OCH3 M N
OH HO-O-CH2
O
HO
O
O
O
HO
O-CH2
P HO-O-CH
HO-O-CH2
100
5. Resultados y discusin
Table 4. Composition of lipophilic extractives (mg/100g) in the different fibers [8, 22-26].
Bast fibers Leaf fibers Reeds
flax hemp kenaf jute sisal abaca curaua giant reed
n-alkanes (A) 27 43 27 5 15 - - 8
fatty alcohols (B) 220 2 13 13 8 <1 55 19
n-aldehydes (C) 371 25 1 - 1 - - 8
fatty acids (D) 552 78 33 13 11 9 81 114
-hydroxyfatty acids (E) - - - 3 6 1 142 -
-hydroxyfatty acids (F) 11 9 - 10 7 1 23 -
free sterols/triterpenols (G) 92 36 5 4 20 25 62 53
sterol/triterpenol esters (H) 6 7 1 - <1 1 9 7
sterol glycosides (I) 5 13 <1 1 2 2 27 15
steroid hydrocarbons (J) 14 30 2 2 14 3 12 13
steroid/triterpenoid ketones (K) 33 27 4 3 3 4 6 4
n-alkyl ferulates (M) - - - - 6 3 - -
ester waxes* (L, N, O, P) 284 17 30 20 8 7 222 42
101
5. Resultados y discusin
Table 5. Composition of H, G and S moieties for all the raw materials studied by Py-GC/MS
[20-25, 35].
Bast fibers Leaf fibers Reeds
flax hemp kenaf jute sisal abaca curaua giant reed
%H 56,4 12.8 1.3 2.1 1.3 20.2 29.8 25.6
%G 40.8 53.0 15.4 32.2 18.7 13.5 29.1 44.2
%S 2.8 34.2 83.3 65.7 80.0 66.2 41.1 30.2
S/G ratio 0.1 0.6 5.4 2.0 4.3 4.9 1.4 0.7
For a more complete structural characterization of the lignins from these non-
woody fibers, the milled-wood lignins (MWL) were isolated and analyzed by
2D-NMR spectroscopy [36-38]. Signals from S and G lignin units were
observed in all spectra, whereas signals for p-hydroxyphenyl (H) lignin units
could only be detected in the HSQC spectra of flax and hemp lignins, in
agreement with the high amounts of these units observed by Py-GC/MS and
shown in Table 5. In general, the relative proportions of the different lignin
units (S/G ratios in Table 6) are in close agreement with the Py-GC/MS data
shown above. In addition, prominent signals corresponding to p-coumarate
structures were observed in the lignins of abaca and curaua [37]. In these
lignins, p-coumaric acid has been reported to be esterified to the lignin polymer
[23, 37, 39]. The side-chain region of the HSQC spectra gave additional
information about the different inter-unit linkages (i.e. -O-4 aryl ether, -
resinol, -5 phenylcoumaran, -1/-O- spirodienones, etc) present in the
structure of these lignins. The main substructures found in these lignins are
depicted in Figure 2. The relative abundances of the main inter-unit linkages
present in the MWL of the non-woody fibers selected for this study are shown in
Table 6. -O-4 aryl ether substructures (I) were predominant in all of the
lignins. Interestingly, the lignins from kenaf, sisal, abaca and curaua are
102
5. Resultados y discusin
especially enriched in -O-4 structures (more than 84% of all side-chains) [37].
Phenylcoumaran (-5 linkages) substructures (II) were observed in most of the
fibers, being especially abundant in flax and hemp, but were completely absent
in abaca. The presence of these low amounts of phenylcoumaran substructures
was expected due to the low levels of guaiacyl lignin units in these samples.
Resinol (- linkages) substructures (III) were also observed in important
amounts in flax, hemp, and jute, and in low amounts in kenaf and sisal, but were
completely absent in abaca and curaua lignins. Finally, spirodienone structures
(IV) were also present, although in lower amounts in most of the fibers, being
absent in flax and hemp. The high abundance of non-condensed linkages in the
lignins of kenaf, sisal, abaca and curaua makes them particularly easily to
delignify, in contrast to the rest of the lignins, with a high content of condensed
linkages, particularly in flax and hemp lignins.
Percentage of -acylation 0 0 58 4 68 80 69
Interestingly, the spectra of some of these lignins (kenaf, sisal, abaca, curaua)
revealed the presence of intense signals corresponding to acylated -carbon
(Figure 2, structures I' and I'') [37]. An estimation of the percentage of -
acylation of the lignin side-chain was calculated by integration of the signals
corresponding to the hydroxylated and acylated -carbon (Table 6) and ranged
from 4% in jute lignin to 80% in abaca lignin. The high level of acylation of the
-carbon has been correlated with the high abundances of -O-4 linkages and
the low abundances of the - resinol structures [37, 38]. The nature of the acyl
group esterifying the -carbon was studied by the so-called Derivatization
Followed by Reductive Cleavage (DFRC) degradation method [40, 41], which
selectively and efficiently cleaves -ether and -ether linkages but leaves -
esters intact. This method allowed confirming that p-coumarate groups are
103
5. Resultados y discusin
E 4 2 E 4 2 E 4' 2'
HO D 3 HO D 3 HO D 3'
O O O
1
OMe 1
OMe 1
OMe
6 2 6 2 6 2
5 3 5 3 5 3
4 4 4
MeO OMe MeO OMe MeO OMe
O O O
I I' I''
1 OMe
6 2
OMe
5 3 O
4 3 O 1''
OMe 2 4
3
6'' 2''
HO 4 3''
E 1 5 2 5''
J O O 6 4''
D J OMe 5 MeO OMe
D MeO 6
1
D
E E
O O
J E D
1 E
6 2 MeO D J
J
5 3 6
1 O HO 1
OH
4 5
MeO 6 2
OMe 4 2
3 5 3
O O 4
MeO OMe
OMe O
II III IV
Figure 2. Main substructures present in the lignins studied here: I, -O-4 linked
substructures; I, -O-4 linked substructures with acetylated -carbon; I, -O-4 linked
substructures; with p-coumaroylated -carbon; II, phenylcoumaran structures formed by -5
and -O-4 linkages; III, resinol structures formed by - , -O-, and -O-R linkages; IV,
spirodienone structures formed by -1, and -O- linkages.
4. Conclusions
The chemical composition of different non-woody plant fibers used as raw
materials for pulp and papermaking has been summarized, with especial
emphasis in the chemistry of lipids and lignin and their fate during alkaline
104
5. Resultados y discusin
pulping. This study offers valuable information that will lead to a better
industrial utilization of these non-woody plant species of high socioeconomic
interest.
Acknowledgements
This study has been supported by the Spanish Projects AGL2005-01748 and
AGL2008-00709 and the EU BIORENEW project (NMP2-CT-2006-26456).
We thank CELESA (Tortosa, Spain) and University of Huelva for providing the
samples. G.M. thanks the Spanish Ministry of Education for a FPI fellowship.
J.R. thanks the Spanish CSIC for a I3P fellowship.
References
[1] Moore G. Nonwood Fibre Applications in Papermaking, Pira International,
Leatherhead, Surrey, UK, 1996.
[2] Paavilainen L. European prospects for using nonwood fibres. Pulp Pap Int
1998; 61-86.
[4] Hurter RW, Riccio FA. Why CEOS dont want to hear about nonwoods-or
should they? In: TAPPI Proceeding, NA Nonwood Fiber Symposium,
Atlanta, GA, USA, 1998; 1-11.
[6] Back EL, Allen LH. Pitch Control, Wood Resin and Deresination, Tappi
press, Atlanta, GA., 2000; pp. 392.
[7] Gutirrez A, del Ro JC. Lipids from flax fibers and their fate after alkaline
pulping. J Agric Food Chem 2003; 51: 4965-4971.
[9] Technical Association of the Pulp and Paper Industry. Test methods, 1992-
1993. TAPPI, Atlanta, Ga. 1993.
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5. Resultados y discusin
[11] Christie WW. In Christie WW, Ed. Solid-phase extraction columns in the
analysis of lipids. Advances in Lipid MethodologyOne, The Oily Press,
Dundee, Scotland 1992; pp. 118.
[15] Capanema EA, Balakshin MY, Chen CL, Gratzl JS, Gracz H. Structural
analysis of residual and technical lignins by 1H-13C correlation 2D NMR-
spectroscopy. Holzforschung 2001; 55: 302-308.
[18] Ralph J, Marita JM, Ralph SA, Hatfield RD, Lu F, Ede RM, Peng J,
Quideau S, Helm RF, Grabber JH, Kim H, Jimenez-Monteon G, Zhang Y,
Jung HJG, Landucci LL, MacKay JJ, Sederoff RR, Chapple C, Boudet
AM. Solution-state NMR of lignin. In: Argyropoulos DS, Ed. Advances in
Lignocellulosics Characterization, Tappi Press, Atlanta, GA 1999; 55-108.
[19] Garca Hortal JA. Fibras Papeleras, Edicions UPC, Barcelona, Spain, 2007;
pp. 243.
106
5. Resultados y discusin
[27] Coelho DS, Marques G, Gutirrez A, Silvestre ARD, del Ro JC. Chemical
characterization of the lipophilic fraction of Giant reed (Arundo donax)
fibres used for pulp and paper manufacturing. Ind Crops Prod 2007; 26:
229-236.
[31] Chen T, Wang Z, Zhou Y, Breui, C, Aschim OK, Yee E, Nadeau L. Using
solid-phase extraction to assess why aspen causes more pitch problems
than softwoods in kraft pulping. Tappi J 1995; 78: 143-149.
107
5. Resultados y discusin
[34] Chang HM, Sarkanen KV. Species variation in lignin. Effect of species on
the rate of Kraft delignification. Tappi press 1973; 56: 132134.
[37] del Ro JC, Rencoret J, Marques G, Gutirrez A., Ibarra D, Santos JI,
Jimnez-Barbero J, Zhang L, Martnez AT. Highly acylated (acetylated
and/or p-coumaroylated) native lignins from diverse herbaceous plants. J
Agric Food Chem 2008; 56: 9525-9534.
[39] Sun RC, Fang JM, Goodwin A, Lawther JM, Bolton J. Fractionation and
characterization of ball-milled and enzyme lignins from abaca fibre. J Sci
Food Agric 1999; 79: 1091-1098.
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5. Resultados y discusin
[43] Ralph J. An unusual lignin from kenaf. J Nat Prod 1996; 59 (4): 341-342.
109
5. Resultados y discusin
Publicacin II:
del Ro J.C., Marques G., Rencoret J. Martnez A.T. and Gutirrez A. (2007)
Occurence of naturally acetylated lignin units. Journal of Agricultural and Food
Chemistry, 55, 5461-5468.
110
5. Resultados y discusin
Jos C. del Ro, Gisela Marques, Jorge Rencoret, ngel T. Martnez, Ana Gutirrez
Instituto de Recursos Naturales y Agrobiologa de Sevilla, CSIC, P.O. Box 1052, 41080-
Seville, Spain
Centro de Investigaciones Biolgicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain
Abstract
In this work, we have studied the occurrence of native acetylated lignin in a
large set of vascular plants, including both angiosperms and gymnosperms, by a
modification of the so-called Derivatization Followed by Reductive Cleavage
(DFRC) method. Acetylated lignin units were found in all angiosperms selected
for this study, including mono- and eudicotyledons, but were absent in the
gymnosperms analyzed. In some plants (e.g. abaca, sisal, kenaf or hornbeam),
lignin acetylation occurred at a very high extent, exceeding 45% of the
uncondensed syringyl lignin units. Acetylation was observed exclusively at the
J-carbon of the lignin-side chain and predominantly on syringyl units, although
a predominance of acetylated guaiacyl over syringyl units was observed in some
plants. In all cases, acetylation appears to occur at the monomer stage and
sinapyl and coniferyl acetates seem to behave as real lignin monomers
participating in lignification.
Keywords: lignin, angiosperms, gymnosperms, eudicotyledons,
monocotyledons, coniferyl acetate, sinapyl acetate, abaca, sisal, kenaf,
hornbeam, Derivatization Followed by Reductive Cleavage (DFRC).
1. Introduction
Lignin is a principal structural component of cell walls in higher terrestrial
plants. In addition to structural support and pathogen defense, lignin functions in
water transport as a hydrophobic constituent of vascular phloem and xylem cells.
Lignins are complex polymers formed by the dehydrogenative polymerization of
three main monolignols, p-coumaryl, coniferyl and sinapyl alcohols.
Gymnosperm lignins are mainly formed from coniferyl alcohol, together with
small proportions of p-coumaryl alcohol. Angiosperm lignins are mainly formed
from coniferyl and sinapyl alcohols with small amounts of p-coumaryl alcohol.
A considerable variation in the contribution of these three alcohol precursors is
observed in lignins from annual plants (1-4). After their synthesis, the lignin
monomers are transported to the cell wall where they are polymerized in a
combinatorial fashion by free-radical coupling mechanisms in a reaction
mediated by peroxidases, generating a variety of structures within the lignin
polymer (5-7).
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5. Resultados y discusin
Some lignins are known to be naturally acetylated at the J-carbon of the side
chain. Acetates have been reported to occur in the lignin of a limited number of
hardwoods and non-woody plants (1, 8, 9). In particular, kenaf bast lignin has
been found to be extensively J-acetylated and predominantly on syringyl units,
although the role of such a highly acetylated lignin is not yet known. Recent
studies have provided strong evidence that sinapyl acetate is implicated as a
monomer in lignification in kenaf bast fibers and that the naturally acetylated
polymeric lignin derives not from acetylation of the lignin polymer but from
polymerization of pre-acetylated monolignols (9, 10). Other acids are also
known to occur naturally acylating lignin; thus, grass lignins are partially p-
coumaroylated and some hardwood lignins such as poplar, aspen or willow are
p-hydroxybenzoylated (7, 11-18).
Naturally acetylated lignins may also occur in other plants but their
occurrence has probably being missed due to the limitations of the analytical
procedures used for their isolation and/or structural characterization. Natural
acetates present on lignin might have been hydrolyzed and removed when using
traditional isolation methods (such as alkaline extraction often applied to non-
wood lignins) and degradative procedures for chemical characterization (such as
nitrobenzene oxidation, CuO oxidation or thioacidolysis). Indeed, for
spectroscopic analysis, e.g. using Nuclear Magnetic Resonance (NMR), lignin is
frequently acetylated for improved solubility and spectroscopic properties and,
therefore, native acetates cannot be seen. Recently, we reported the occurrence
of acetylated lignins in some herbaceous plants, including kenaf, jute, sisal and
abaca, characterized by a high syringyl (S) to guaiacyl (G) ratio, by the use of
Pyrolysis coupled to Gas Chromatography-Mass Spectrometry (Py-GC/MS)
(19-21), although the method used hindered the determination of the extent of
acetylation. In this work we have studied the occurrence and abundance of
native acetylated lignin units in the milled wood lignins (MWL) isolated from a
wide set of vascular plants, including gymnosperms and angiosperms (mono-
and eudicotyledons). For this purpose, we have used a modification of the so-
called Derivatization Followed by Reductive Cleavage (DFRC) degradation
method (22-24). DFRC is a simple and powerful method which selectively and
efficiently cleaves D-ether and E-ether linkages and allows quantitative analysis
of structural units in etherified lignin, and also provides some information on
carbon-carbon linked lignin by analysis of the dimeric structures released.
DFRC includes two key steps, (i) solubilization of lignin by bromination and
acetylation with acetyl bromide and (ii) reductive cleavage of the aryl ether
bonds in lignin with zinc dust. Identification of the resulting monomeric and
dimeric degradation products by GC/MS gives valuable information on the
lignin structure. However, and most importantly, DFRC leaves J-esters intact
allowing the analysis of native J-acylated lignin. Thus, the method has allowed
to confirm that p-coumarate groups are attached at the J-carbon of grass lignins,
predominantly on syringyl units (17). However, the DFRC method uses
112
5. Resultados y discusin
acetylating reagents that interfere in the analysis of native acetates in lignin, but
with appropriate modification by substituting acetylation by propionylation (25),
it is possible to obtain significant information and clues about the occurrence
and extent of native lignin acetylation. In this paper, we use this method to
investigate the presence of naturally acetylated lignin units in a set of vascular
plants, including angiosperms and gymnosperms.
2.2. DFRC
A modification of the standard DFRC method by using propionyl instead of
acetyl reagents (DFRC) was used (25). Lignins (10 mg) were stirred for two
hours at 50 C with propionyl bromide in propionic acid (8:92, v/v). The
solvents and excess of bromide were removed by rotary evaporation. The
products were then dissolved in dioxane/propionic acid/water (5:4:1, v/v/v), and
50 mg powered Zn was added. After 40 min stirring at room temperature, the
mixture was transferred into a separatory funnel with dichloromethane and
saturated ammonium chloride. The aqueous phase was adjusted to pH < 3 by
adding 3% HCl, the mixture vigorously mixed and the organic layer separated.
The water phase was extracted twice more with dichloromethane. The combined
dichloromethane fractions were dried over anhydrous NaSO4 and the filtrate was
evaporated in a rotary evaporator. The residue was subsequently propionylated
113
5. Resultados y discusin
114
Table 1. Abundance (Molar Yields) of the DFRC Degradation Monomers of the MWL Isolated from the Different Plants Selected for
This Study, S/G Ratios and Relative Abundances of Acetylated Lignin Moieties.
Eudicotyledons
Fagales Fagaceae Fagus sylvatica Beech 126 2 165 20 1.4 10.8 1.6
Fagales Betulaceae Carpinus betulus Hornbeam 146 4 230 185 2.8 44.6 2.7
Rosales Cannabaceae Cannabis sativa Hemp 286 2 177 2 0.6 1.1 0.7
Malvales Malvaceae Hibiscus cannabinus Kenaf 390 38 543 780 3.1 59.0 8.9
Malvales Malvaceae Corchorus capsularis Jute 299 1 336 23 1.2 6.4 0.3
Malpighiales Salicaceae Populus tremula Aspen 651 5 662 8 1.0 1.2 0.8
Myrtales Myrtaceae Eucalyptus globulus Eucalypt 154 8 275 3 2.3 1.1 4.9
Gymnosperms
Coniferales Pinaceae Picea abies Spruce 520 0 0 0 0.0 - 0.0
a
%Sac is the percentage of acetylated S units respect to the total S units. b %Gac is the percentage of acetylated G units respect to the total G units. cSome amounts of
J-p-coumaroylated S units were found (27 an 11 Pmol/g lignin for bamboo and abaca, respectively) and were included in the estimation of total S units for calculation
of S/G and %Sac.
5. Resultados y discusin
115
5. Resultados y discusin
predominance of the trans- over the cis- form, as also occurred with the
corresponding non-acetylated alcohols.
The results from the DFRC analysis of the MWL selected for this study,
namely the molar yields of the released monomers, the S/G ratios and the
percentages of naturally acetylated guaiacyl (%Gac) and syringyl (%Sac) lignin
moieties, are presented in Table 1. The yields of the released monomers were in
the same range as previously observed in the DFRC degradation of other
isolated lignins (17, 24). As shown in Table 1, naturally acetylated lignin units
were found to occur in all angiosperms analyzed in the present study, including
both mono- and eudicotyledons. However, no traces of acetylated lignin units
could be found in the MWL of the two gymnosperms (pine and spruce) studied
here. The data also indicated that in most lignin samples acetylation occurred
predominantly on syringyl units, whereas only traces of acetylated guaiacyl
units were detected, although in bamboo and eucalyptus lignins, with a low
extent of acetylation, this occurred preferentially on guaiacyl units. We can
exclude acetylation as an artifact produced during the lignin isolation protocol
since MWL from pine and spruce (where no traces of acetylated units could be
detected) were also isolated using the same procedure as the rest of the samples.
Indeed, acetates were found predominantly on S lignin units and exclusively at
the J-carbon, which suggests that they are naturally present. And finally, direct
DFRC of some whole fibers (such as sisal and kenaf), without previous lignin
isolation, gave also similar results.
The occurrence of naturally acetylated lignin units seems to be widespread
among angiosperms and restricted only to this group of vascular plants, being
particularly abundant in syringyl-rich lignins. Especially important is the high
extent of lignin acetylation observed in the MWL from the herbaceous plants
abaca, sisal and kenaf, and in the hardwoods hornbeam and, in a minor extent,
beech, all of them characterized by high S/G ratios. However, we also noted the
acetylation of S units in coir lignin, which is characterized by a very low S/G
ratio (0.2). The high extent of acetylation of kenaf lignin has been previously
reported by NMR and DFRC (1, 25). The occurrence of naturally acetylated
lignin units was also reported in kenaf, jute, sisal and abaca by Py-GC/MS of the
whole fibers, although the extent of acetylation could not be determined due to
the limitations of the technique (19-21). Interestingly, the high extent of
acetylation of sisal MWL included both S units (78%) and G units (50%),
whereas in the case of abaca, kenaf or hornbeam lignins, acetylation occurred
almost predominantly on S units (80, 59 and 45%, respectively) and only a
minor degree of acetylation was observed on G units (6, 9 and 3%, respectively).
116
5. Resultados y discusin
t-Sac
100
Sisal
relative intensity
t-G
t-G ac t-S
c-G
c-Sac c-S
c-G ac
0
t-Sac
100
t-S
Kenaf
t-G
relative intensity
t-G ac
c-G ac
0
t-S
100
t-Sac
Hornbeam
t-G
relative intensity
c-Gt-G ac c-S
c-Sac
0
t-Sac
100
Abaca
relative intensity
t-G
t-S
c-Sac
c-G
t-G ac c-S
0
10 15 20
Retention time (min)
Figure 1. Chromatograms of the DFRC degradation products of selected MWL from sisal,
kenaf, hornbeam and abaca. c-G, t-G, c-S and t-S are the cis- and trans-guaiacyl and syringyl
monomers, respectively. c-Gac, t-Gac, c-Sac and t-Sac are the originally acetylated cis- and
trans-guaiacyl and syringyl monomers, respectively.
On the other hand, it has been recently and elegantly proved that acetylated
lignin in kenaf derives from polymerization of pre-acetylated monolignols and
not from acetylation of the lignin polymer (9, 10). Acetylation of lignin at
monomer stage only partially affects the course of the coupling reactions, and
117
5. Resultados y discusin
OProp OProp
OCH 3 CH 3O OCH3
OProp OProp
G S
M+= 292; [M+-56]= 236 M+= 322; [M+-56]= 266
O O
=
=
O-C-CH3 O-C-CH 3
Gac Sac
M+= 278; [M+-56]= 222 M+= 308; [M+-56]= 252
Figure 2. Structures and mass fragments of the J-OH (G and S) and naturally J-acetylated
(Gac and Sac) lignin monomers released after DFRC of MWL.
118
5. Resultados y discusin
O O
=
O-C-CH3 O-C-CH 3 O O
J J
=
J J
CH3-C-O O-C-CH 3
E E
D D E E
EE D D
CH3O OCH3
O
+ homo-coupling
HO OH
CH 3O OCH3 CH3O OCH3
OH OH OCH 3 I OCH3
OH
O CH3O OCH3
=
OH O-C-CH 3
J J
E E J
D D E
D
EE HO
O
J
+ cross-coupling O E D
=
CH 3-C-O
CH3O OCH3 CH3O OCH3
OCH3
OH OH
CH 3O OH
II
OCH3
OH
OH OH
J J O
J D OCH3
E E
D D E E
EE D J
CH3O O
+ homo-coupling
HO III
CH3O OCH3 CH3O OCH3
OH OH OCH3
Figure 3. Structures of the tetrahydrofuran dimers arising from the EE coupling of sinapyl
alcohol and sinapyl acetate. I: EEcoupling product of two sinapyl acetates; II: EEcoupling
product of a sinapyl alcohol and a sinapyl acetate; III: EEcoupling product (syringaresinol)
of two sinapyl alcohols.
119
5. Resultados y discusin
120
5. Resultados y discusin
CH 3O O
=
O-C-CH 3
O-C-CH 3
=
PrO
O
DFRC
I CH 3O
CH 3O OCH3
OPr
CH 3O CH 3O O
=
OPr O-C-CH3
O-C-CH 3 OPr
=
PrO PrO
O
DFRC
II CH 3O + CH3O
CH 3O OCH3 CH 3O OCH 3
OPr OPr
IIa; M+= 630; [M-56]+= 574 IIb; M+= 630: [M-56]+= 574
CH 3O
OPr
OPr
DFRC PrO
III
CH3O
CH 3O OCH 3
OPr
Figure 4. Aryltetralin products resulting from the DFRC degradation of the di- (I), mono-
(IIa and IIb) and none-acetylated (III) EE coupling structures. The molecular mass and
base peaks are indicated under the structures.
Although it is now evident that native acetylated lignin units are widespread,
and probably ubiquitous, in angiosperms, the role of such lignin acetylation in
the plant is not yet known. Some studies indicated that J-acylation with p-
coumarates may function as radical transfer carriers to help sinapyl alcohol
incorporate into lignin when the wall peroxidases have a low reactivity with
sinapyl alcohol directly (28). However, this is not the case for acetylated lignin
monomers and the function of such acetylated lignin remains unknown.
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5. Resultados y discusin
IIa,IIb
100
Sisal
III
relative intensity
I
0
III
100
Kenaf
relative intensity
IIa,IIb
0
III
100
Hornbeam
relative intensity
IIa,IIb
0
100
Abaca
relative intensity
0
35 36 37 38 39 40
Retention time (min)
Figure 5. Detail of reconstructed (sum of the ions at m/z 560, 574 and 588) chromatograms of
the DFRC degradation products of selected MWL from sisal, kenaf, hornbeam and abaca,
showing the presence of aryltetralin EE products containing two (I), one (II and IIb) and
none (III) native acetates.
The high extent of lignin acetylation of sisal and other Agaves (preliminary
results on another species, Agave americana, by DFRC of the whole fiber,
without previous lignin isolation, also indicate more than 75% of acetylated S
units) can provide some answers about its role in this plant. Since the resultant
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5. Resultados y discusin
acetylated lignin polymer is more hydrophobic than normal lignin, the role of
lignin acetylation could be associated with drought tolerance, as already
advanced by Ralph (29). Sisal, as other Agaves, is a drought tolerant desert
perennial plant successfully copying with high temperatures and desiccation.
Agaves are characterized by tissue succulence and Crassulacean Acid
Metabolism to minimize water loss. The presence of a highly acetylated lignin
in Agaves will increase hydrophobicity of the vascular tissues thus helping to
reduce water loss in the plant. However, abaca, despite being highly acetylated,
has not succulent leaves and is not drought tolerant, and therefore the highly
acetylated lignin might have a different role for this plant. In this case, the role
of the high extent of lignin acetylation could be more related with the lower
affinity of acetylated monolignols to form EE linkages producing a more non-
condensed lignin enriched in E-O-4 linkages. Whatever the reason for the
occurrence of acetylated lignin in plants, it seems that the mechanism for lignin
acetylation confers the plant a high flexibility to produce different types of
lignins with different degree of acetylation to adapt to different environmental
conditions.
In conclusion, the presence of naturally acetylated lignin units, which in some
plants make up to 80% of the uncondensed S lignin, has been largely
underestimated. This has mainly been due to the analytical methodologies used
for their isolation and structural characterization, which are not appropriate for
the analysis of native acetylated lignin. Therefore, all subsequent lignin
structural studies should take into account the occurrence of these moieties and
accordingly adapt the methodological protocols used.
Acknowledgements
This study has been supported by the Spanish MEC (project AGL2005-01748)
and EU contract NMP2-CT-2006-26456. JR thanks the Spanish CSIC for an I3P
fellowship; GM thanks the Spanish Ministry of Education for a FPI fellowship.
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Fractionation and characterization of ball-milled and enzyme lignin from
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linkages between hydroxycinnamic acids and lignin from wheat, rice, rye,
and barley straws, maize stems, and fast-growing poplar wood. Ind. Crops
& Prod. 2002, 15, 179-188.
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[4] del Ro, J.C.; Gutirrez, A.; Rodrguez, I.M., Ibarra, D., Martnez, A.T.
Composition of non-woody plant lignins and cinnamic acids by Py-
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Biol. 2003, 54, 519546
[7] Ralph, J.; Lundquist, K.; Brunow, G.; Lu, F.; Kim, H.; Schatz, P. F.;
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Lignins: natural polymers from oxidative coupling of 4-
hydroxyphenylpropanoids. Phytochem. Rev. 2004, 3, 29-60.
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[10] Lu, F.; Ralph, J. Novel EEstructures in lignins incorporating acylated
monolignols. In Proceedings of 13th International Symposium on Wood,
Fiber, and Pulping Chemistry, Auckland, New Zealand, May 16-19, 2005;
Volume 3, pp. 233-237.
[11] Smith, D. C. C. p-Hydroxybenzoate groups in the lignin of aspen (Populus
tremula). J. Chem. Soc. 1955, 23472351.
[12] Nakano, J.; Ishizu, A.; Migata, N. Studies on lignin. XXXII. Ester groups
of lignin. Tappi 1961, 44, 3032.
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(Recent data on the heterogeneity of lignin). Physiol. Veg. 1981, 19, 327-
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[14] Landucci, L. L.; Deka, G. C.; Roy, D. N. A. 13C NMR study of milled
wood lignins from hybrid Salix clones. Holzforschung 1992, 46, 505-511.
[15] Ralph, J.; Hatfield, R. D.; Quideau, S.; Helm, R. F.; Grabber, J. H.; Jung,
H. -J. G. Pathway of p-coumaric acid incorporation into maize lignin as
revealed by NMR. J. Am. Chem. Soc. 1994, 116, 9448-9456.
[16] Sun, R. C.; Fang, J. M.; Tomkinson, J. Fractional isolation and structural
characterization of lignins from oil palm trunk and empty fruit bunch
fibres. J. Wood Chem. Technol. 1999, 19, 335356.
[17] Lu, F. and Ralph, J. Detection and determination of p-coumaraloylated
units in lignin. J. Agric. Food Chem. 1999, 47, 1985-1992.
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[18] Meyermans, H.; Morreel, K.; Lapierre, C.; Pollet, B.; De Bruyn, A.;
Busson, R.; Herdewijn, P:; Devreese, B.; Van Beeumen, J.; Marita, J. M.;
et al. Modification in lignin and accumulation of phenolic glucosides in
poplar xylem upon down-regulation of caffeoyl-coenzyme A O-
methyltransferase, an enzyme involved in lignin biosynthesis. J. Biol.
Chem. 2000, 275, 3689936909.
[19] del Ro, J. C.; Gutirrez, A.; Martnez A. T. Identifying acetylated lignin
units in non-wood fibers using pyrolysis-gas chromatography/mass
spectrometry. Rapid Commun. Mass Spectrom. 2004, 18, 1181-1185.
[20] Gutirrez, A.; Rodrguez, I. M.; del Ro J. C. Chemical characterization of
lignin and lipid fractions in kenaf bast fibers used for manufacturing high-
quality papers. J. Agric. Food Chem. 2004, 52, 4764-4773.
[21] del Ro, J. C.; Gutirrez, A. Chemical composition of abaca (Musa textilis)
leaf fibers used for manufacturing of high quality paper pulps. J. Agric.
Food Chem. 2006, 54, 4600-4610.
[22] Lu, F.; Ralph, J. Derivatization followed by reductive cleavage (DFRC
method), a new method for lignin analysis: protocol for analysis of DFRC
monomers. J. Agric. Food Chem. 1997, 45, 2590-2592.
[23] Lu, F.; Ralph, J. The DFRC method for lignin analysis. Part 1. A new
method for Earyl ether cleavage: lignin model studies. J. Agric. Food
Chem. 1997, 45, 4655-4660.
[24] Lu, F.; Ralph, J. The DFRC method for lignin analysis. 2. Monomers from
isolated lignin. J. Agric. Food Chem. 1998, 46, 547-552.
[25] Ralph, J.; Lu, F. The DFRC method for lignin analysis. 6. A simple
modification for identifying natural acetates in lignin. J. Agric. Food Chem.
1998, 46, 4616-4619.
[26] Bjrkman, A. Studies on finely divided wood. Part I. Extraction of lignin
with neutral solvents. Sven. Papperstidn. 1956, 59, 477-485.
[27] Sederoff, R. D.; MacKay, J.; Ralph, J.; Hatfield, R. Unexpected variation in
lignin. Curr. Opin. Plant Biol. 1999; 2: 145.
[28] Takahama, U.; Oniki, T.; Shimokawa, H. A possible mechanism for the
oxidation of sinapyl alcohol by peroxidase-dependent reactions in the
apoplast: enhancement of the oxidation by hydroxycinnamic acids and
components of the apoplast. Plant Cell Physiol. 1996, 37, 499-504.
[29] Ralph, J. Elucidation of new pathways in normal and perturbed
lignification. Appita 2005, 3-13.
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Publicacin III:
del Ro J.C., Rencoret J., Marques G., Gutirrez A., Ibarra D., Santos J.I.,
Jimnez-Barbero J., Zhang L. and Martnez A.T. (2008) Highly acylated
(acetylated and/or p-coumaroylated) native lignins from diverse herbaceous
plants. Journal of Agricultural and Food Chemistry, 56, 9525-9534.
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Instituto de Recursos Naturales y Agrobiologa de Sevilla, CSIC, P.O. Box 1052, 41080-
Seville, Spain
Centro de Investigaciones Biolgicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain
Royal Institute of Technology (KTH), Fiber and Polymer Technology, SE-100 44
Stockholm, Sweden
Abstract
The structure of lignins isolated from the herbaceous plants sisal (Agave
sisalana), kenaf (Hibiscus cannabinus), abaca (Musa textilis) and curaua
(Ananas erectifolius) has been studied upon spectroscopic (2D-NMR) and
chemical degradative (Derivatization Followed by Reductive Cleavage)
methods. The analyses demonstrate that the structure of the lignins from these
plants is highly remarkable, being extensively acylated at the -carbon of the
lignin side-chain (up to 80% acylation) with acetate and/or p-coumarate groups,
and preferentially over syringyl units. While the lignins from sisal and kenaf are
-acylated exclusively with acetate groups, the lignins from abaca and curaua
are esterified with acetate and p-coumarate groups. The structures of all these
highly-acylated lignins are characterized by a very high syringyl/guaiacyl ratio,
a large predominance of -O-4 linkages (up to 94% of all linkages) and a
strikingly low proportion of traditional -linkages, which indeed are
completely absent in the lignins from abaca and curaua. The occurrence of -
homo-coupling and cross-coupling products of sinapyl acetate in the lignins
from sisal and kenaf indicates that sinapyl alcohol is acetylated at monomer
stage and that, therefore, sinapyl acetate should be considered as a real
monolignol involved in the lignification reactions.
Keywords: lignin, herbaceous plants, sinapyl acetate, sinapyl p-coumarate, 2D-
NMR, HSQC, DFRC, sisal, kenaf, abaca, curaua
1. Introduction
Lignins are complex natural biomacromolecules characteristics of vascular
plants, where they provide mechanical support. In addition, lignin waterproofs
the cell wall, enabling transport of water and solutes through the vascular
system, and plays a role in protecting plants against pathogens (1). The lignin
polymer results from the random oxidative coupling of p-hydroxycinnamyl
monolignols mediated by laccases and/or peroxidases (2, 3). The three primary
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5. Resultados y discusin
128
5. Resultados y discusin
129
5. Resultados y discusin
130
5. Resultados y discusin
131
5. Resultados y discusin
GC GC
(a) MeO CE (b) MeO CE
55 55
DE DE
AJ AJ
A' J 60 A' J 60
65 65
AD/AD CJ 70 AD/AD CJ 70
75 75
DD carbohydrates 80 DD 80
A' E A' E carbohydrates
D D CD
AE D D CD AE
85 85
GH 5.0 4.5 4.0 3.5 3.0 GH 5.0 4.5 4.0 3.5 3.0
GC GC
(c) MeO (d) MeO
DE 55 55
AJ AJ
A' J/A'' J 60 A' J/A'' J 60
65 65
AD/AD/AD 70 AD/AD/AD 70
75 75
carbohydrates
DD A' E/A'' E 80 DD carbohydrates 80
A' E/A'' E
D D AE D D AE
85 85
GH 5.0 4.5 4.0 3.5 3.0 GH 5.0 4.5 4.0 3.5 3.0
Figure 1. Expanded side-chain region, GC/GH 50-90/2.5-5.5 ppm, of the HSQC spectra of the
lignins from (a) sisal, (b) kenaf, (c) abaca and (d) curaua. Carbohydrate signals are presented
in grey color. See Table 1 for signal assignment and Figure 3 for the main lignin structures
(A-D) identified.
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5. Resultados y discusin
GC GC
(a) S2,6 100
(b) S2,6 100
S2,6 (CD=O) S2,6 (CD=O)
S/G 3.9 S/G 5.6
D 2` 110 D 2` 110
G2 G2
G5 D 6` G5
D 6`
G6 120 G6 120
130 130
140 140
GH 8.0 7.5 7.0 6.5 6.0 GH 8.0 7.5 7.0 6.5 6.0
GC GC
(c) S 2,6 100 (d) S2,6 100
S2,6 (CD=O) S 2,6 (CD=O)
S/G 8.7 S/G 4.9
D 2` 110 D 2` 110
G2 A'' E G2 A'' E
G5 G5
G6 D 6` D 6` 120
120 G6
A'' 3,5 A'' 3,5
A'' 2,6 A'' 2,6
130 130
GH 8.0 7.5 7.0 6.5 6.0 GH 8.0 7.5 7.0 6.5 6.0
Figure 2. Expanded aromatic region, GC/GH 95-150/5.5-8.5 ppm, of the HSQC spectra of the
lignins from (a) sisal, (b) kenaf, (c) abaca and (d) curaua. See Table 1 for signal assignment
and Figure 3 for the main lignin structures (A-D) identified. G and S are the guaiacyl and
syringyl aromatic units, respectively.
The side-chain region of the HSQC spectra gives also additional information
about the inter-unit linkages present in the structure of these lignins. All the
spectra showed prominent signals corresponding to EO-4 aryl ether linkages.
The CD-HD correlations in EO-4 substructures were observed at GC/GH
72.3/4.86 ppm (structures A, A' and A''), while the CE-HE correlations were
observed at GC/GH 86.5/4.10 ppm in normal J-OH EO-4 aryl ether
substructures (A) but shifted to GC/GH 83.6/4.32 ppm in J-acylated EO-4 aryl
ether substructures (A', A''). EO-4 aryl ether substructures were highly
predominant in all the lignins analyzed here although other substructures were
also observed. Small signals corresponding to spirodienone (E1, DOD
linkages) substructures (D) can be observed in the spectra of sisal, kenaf, abaca
and curaua lignins. Signals of spirodienone CD-HD, CD-HD and CE-HE
correlations were observed at GC/GH 85.4/4.64, 85.4/4.80 and 56.1/3.09 ppm,
respectively. Spirodienone substructures were previously reported in the lignin
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5. Resultados y discusin
13
Table 1. Assignment of Main Lignin C-1H Cross-Signals in the MWL HSQC Spectra
Shown in Figures 1 and 2.
C/H (ppm) Assignment
53.7/3.12 C-H in EE' (resinol) substructures (C)
56.1/3.09 C-H in E'(spirodienone) substructures (D)
60.0/3.38-3.71 CH in -O-4' substructures (A)
63.8/3.83-4.30 CH in J-acylated -O-4' substructures (A' and A'')
71.7/3.81 and 4.17 C-H in EE' (resinol) substructures (C)
72.3/4.86 C-H in -O-4' substructures (A, A' and A'')
82.1/5.12 CD-HD in E'(spirodienone) substructures (D)
83.6/4.32 C-H in J-acylated -O-4' substructures (A' and A'')
85.4/4.64 C-H in EE' (resinol) substructures (C)
85.4/4.80 CD-HD in E'(spirodienone) substructures (D)
86.5/4.10 C-H in J-OH -O-4' substructures (A)
87.7/5.45 CD-HD in phenylcoumaran substructures (B)
103.8/6.68 C2-H2 and C6-H6 in syringyl units
106.7/7.36 and 7.21 C2-H2 and C6-H6 in oxidized (C=O) syringyl units
111.5/6.99 C2-H2 in guaiacyl units
111.6/6.23 C2-H2 in E'(spirodienone) substructures (D)
114.3/6.24 CE-HE in p-coumaroylated substructures (A'')
115.2/6.71 and 6.94 C5-H5 in guaiacyl units
116.2/6.77 C3-H3 and C5-H5 in p-coumaroylated substructures (A'')
118.3/6.19 C6-H6 in E(spirodienone) substructures (D)
119.5/6.83 C6-H6 in guaiacyl units
130.5/7.4 C2-H2 and C6-H6 in p-coumaroylated substructures (A'')
145.1/7.39 CD-HD in p-coumaroylated substructures (A'')
from kenaf bast fibers by Zhang et al. (37). Phenylcoumaran (E5 linkages)
substructures (B) were also found, although in very small proportions. Very
weak signals corresponding to CD-HD correlations of phenylcoumaran
substructures at GC/GH 87.7/5.45 ppm were observed in the spectra of sisal, kenaf
and curaua lignins, but were absent in the spectrum of abaca. The presence of
these low amounts of phenylcoumaran substructures was expected due to the
very low levels of guaiacyl lignin units in all these samples. Finally, resinol
(EE linkages substructures (C) were clearly observed in the spectrum of
kenaf. Signals for the CD-HD, CE-HE and the double CJ-HJ correlations of resinol
substructures were observed at GC/GH 85.4/4.64, 53.7/3.12 and 71.7/3.81 and
4.17 ppm, respectively. Resinol substructures could also be observed, although
in very small traces, in the spectrum of sisal, but were completely absent in the
spectra of abaca and curaua lignins. The relative abundances of the main inter-
unit linkages present in the MWL selected for this study were calculated from
the HSQC spectra and are shown in Table 2. All these highly acetylated lignins
share a common characteristic, the strikingly high proportion of EO-4 ether
linkages (up to 94% of all linkages) and a very low proportion of condensed
linkages (i.e. E, E and EE). Some of these condensed linkages (E
and EE) are even absent in some lignins (abaca and curaua).
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5. Resultados y discusin
The main cross-signals in the aromatic region of the HSQC spectra (Figure 2)
correspond to the aromatic rings of the different lignin units. Signals from
syringyl- (S) and guaiacyl- (G) lignin units can be observed in all spectra. The
syringyl units show a prominent signal for the C2,6-H2,6 correlation at GC/GH
103.8/6.68 ppm, while guaiacyl units showed different correlations for C2-H2
(GC/GH 111.5/6.99 ppm), C5-H5 (GC/GH 115.2/6.71 and 6.94) and C6-H6 (GC/GH
119.5/6.83 ppm). Signals corresponding to C2,6-H2,6 correlations in CD-oxidized
S-lignin units were observed at GC/GH 106.7/7.36 and 7.21 ppm. No signals for p-
hydroxyphenyl (H) lignin units could be detected in the HSQC spectra of these
lignins. An estimation of the relative proportions of the S and G-lignin units in
the HSQC spectra revealed that all the lignins selected for this study present a
very high S/G ratio, ranging from 3.9 in sisal to 8.7 in abaca (Table 2). Other
signals present in this region of the HSQC spectra are from spirodienone
substructures (D) with C2-H2 and C6-H6 correlations at GC/GH 111.6/6.23 and
118.3/6.19, respectively. Prominent signals corresponding to p-coumarate
structures were observed in the lignins of abaca and curaua. Cross-signals
corresponding to the correlations C2,6-H2,6 at GC/GH 130.5/7.40 ppm and C3,5-
H3,5 at GC/GH 116.2/6.77 ppm of the aromatic ring and signals for the
correlations of the unsaturated CD-HD at GC/GH 145.1/7.39 and CE-HE at
114.3/6.24 ppm of the p-coumarate unit in structure A'' of Figure 3, were
observed in this region of the HSQC spectra of abaca and curaua. In abaca
lignin, p-coumaric acid has already been reported to be esterified to the lignin
polymer (8, 16, 35, 38).
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5. Resultados y discusin
the nature of the acyl group (besides the occurrence of acetate and p-coumarate
moieties). A sensitive and selective method is therefore needed to reveal the
HO
5
4 6
3 1
O 2 O
E
D J
1
OMe 1
OMe OMe
1
6 2 6 2 6 2
5 3 5 3 5 3
4 4 4
MeO OMe MeO OMe MeO OMe
O O O
A A' A''
1 OMe
6 2
OMe
5 3 O
4 3 O 1''
OMe 2 4 6'' 2''
3
HO 4
1 5 5'' 3''
E O 2
J O 6 4''
D J OMe 5 MeO OMe
D MeO 1
6
D
E E
O O
1 J E D
E
6 2 D J
MeO J
5 3 6 HO OH
5 1 O 1
4
6 2
MeO OMe 4 2
3 5 3
O O 4
MeO OMe
OMe O
B C D
Figure 3. Main structures present in the highly acylated lignins studied here: (A) E2-4 aryl
ether linkages; (A') E2-4 aryl ether linkages with acetylated J-carbon; (A'') E2-4 aryl
ether linkages with p-coumaroylated J-carbon; (B) phenylcoumaran structures formed by
E5 and D2-4 linkages; (C) resinol structures formed by EE, DOJand JOD
linkages; and (D) spirodienone structures formed by E1,DO-D linkages.
nature of the acyl group that is esterifying the J-carbon of the lignin side-chain
and to know to which lignin moiety it is attached. The DFRC degradation
method, which cleaves D- and E-ether linkages in the lignin polymer leaving J-
esters intact (26-28), seems to be the most appropriate method for the analysis of
native J-acylated lignin.
DFRC analysis of the lignin samples selected for this study allowed
confirming that p-coumarate groups are attached to the J-carbon of abaca and
curaua lignins, and predominantly on syringyl units (Figure 4). Saturated p-
coumarate (dihydro-p-coumarate) esterified to sinapyl alcohol (as its acetate
derivative, Sdpc) was expected to be the major DFRC degradative compound,
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5. Resultados y discusin
according to Lu and Ralph (9), and this was the only degradation product that
was quantified in our previous paper (16). However, a closer look to other major
degradation compounds produced upon DFRC of abaca and curaua lignins
indicated the release of important amounts of the unsaturated counterpart, that
is, intact sinapyl p-coumarate (as its acetate derivative, Spc), that was biased, and
therefore not quantified, in our previous paper. Therefore, in this work, we have
now taken into account both compounds to quantify the total abundance of
sinapyl p-coumarate units present in these lignins (Table 3). Trace amounts of
the respective coniferyl p-coumarate could also de detected in abaca and curaua
lignins. Moreover, some amounts of free p-coumaric acid (as its acetate
derivative) could be observed among the DFRC degradation products of curaua
lignin as a broad peak (Figure 4), that could probably correspond to p-coumaric
acid moieties linked to lignin through EO-4 aryl-ether bonds.
The original DFRC degradation method, however, does not allow the analysis
of native acetylated lignin because the degradation products are acetylated
during the degradation procedure, but with appropriate modification of the
protocol by substituting acetylating reagents with propionylating reagents,
DFRC, it is also possible to obtain information about the occurrence of native
lignin acetylation (13). Figure 5 shows the chromatograms of the DFRC
products released from the lignin samples selected in this study. All the analyzed
lignins released the cis and trans isomers of guaiacyl (c-G and t-G) and syringyl
(c-S and t-S) lignin monomers (as their propionylated derivatives) arising from
normal J-OH units in lignin. In addition, the presence of originally J-acetylated
guaiacyl (c-Gac and t-Gac) and syringyl (c-Sac and t-Sac) lignin units could also be
clearly observed in the chromatograms of all of the selected lignins indicating
that acetylation occurred exclusively at the J-carbon of the lignin side-chain, as
already observed in the HSQC spectra.
Table 3. Abundance (Molar Yields) of the DFRC and DFRC degradation monomers of the
MWL isolated from the different plants selected for this study, and relative abundances of the
different acylated (acetylated and p-coumaroylated) lignin moieties.
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5. Resultados y discusin
100 tS
(a)
relative intensity
Spc
tG
cS
Spc
S
Sdpc pc
0
100 tS
(b)
relative intensity
Spc
tG
pC
cS Spc
Sdpc Spc
0
5 10 15 20 25
Retention time (min)
Figure 4. Chromatograms of the DFRC degradation products of the MWL from (a) abaca and
(b) curaua, showing the presence of sinapyl alcohol esterified to p-coumarate moieties. Sdpc
and Spc are the sinapyl alcohol esterified with dihydro-p-coumarate and p-coumarate,
respectively (as their acetyl derivative). c-G, t-G, c-S and t-S are the normal cis- and trans-
guaiacyl and syringyl monomers, respectively, (as their acetyl derivatives).
The results from the DFRC and DFRC analysis of the MWL selected for this
study, namely the molar yields of the released monomers, the percentages of
naturally acetylated guaiacyl (%Gac) and syringyl (%Sac) and p-coumaroylated
guaiacyl (%Gpc) and syringyl (%Spc) lignin moieties, and the S/G ratios, are
presented in Table 3. The data indicate that a high extent of J-acetylation occurs
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5. Resultados y discusin
in all lignins studied here, and that p-coumaric acid is also found partially
esterifying the lignin of abaca and curaua, in agreement with the NMR data. In
all cases, acetate and p-coumarate groups are preferentially attached to syringyl
units, as previously noted for other lignins (7-9, 12-16, 39). Interestingly, the
high extent of acetylation observed in sisal and curaua also included the G-lignin
units (around 50% of acetylation in both cases). By contrast, in kenaf and abaca
lignins, the J-carbon of G-lignin units is mostly not esterified. On the other
hand, the high extent of acylation of the lignin monomers observed by the
DFCR (and DFRC) method, which can only analyze non-condensed lignin
moieties, is in accordance with the high extent of lignin acylation observed by
HSQC technique, which allows the analysis of the entire MWL structure,
including both condensed and non-condensed linkages. This fact indicates that
both condensed and non-condensed moieties have similar extent of acylation.
However, we must now convey again that the results presented here reflect only
the structure of the isolated MWL, which only represent a small part of the
entire native lignin. However, similar lignin S/G ratio and acylation degree have
been found by in situ analysis in HSQC spectra of the whole cell wall material
(without lignin isolation) at the gel state (40), indicating that MWL can still be
considered as the most representative preparation for the plant native lignin, in
spite of its low yield.
Previous papers describing the structure of some of these lignins have failed
to detect their high levels of acetylation. A recent paper describing the structure
of sisal lignin (41) did not detect the high levels of acetylation, despite of using
spectroscopic techniques. Probably, this was due to the method used for
isolation (acidolysis) that might have hydrolyzed the acetyl groups, or to a
misassignment of the spectral bands. Previous structural studies on abaca lignin
(8, 16, 36, 38), using different degradation methods, also suggested the
occurrence of p-coumaroylated units attached to the J-carbon of the lignin side-
chain. The presence of acetylated J-carbons was also observed in abaca fibers
directly by Py-GC/MS (15) although other authors failed to detect their presence
(8).
On the other hand, the question to whether acylated lignin derives from
polymerization of acylated monolignols or from acylation of the lignin polymer
has recently been addressed and sinapyl acetate has been demonstrated to
behave as a monomer in lignification participating in coupling reactions (14, 16,
17, 42). Part of the evidence comes from the coupling reactions. If the -
carbon of a monolignol is pre-acylated, the formation of the normal resinol
structures can not occur because the absence of free -hydroxyls needed to re-
aromatize the quinone methide moiety. Instead, new tetrahydrofuran structures
are formed from the EE homo- and cross-coupling of two sinapyl (acylated
and non-acylated) monolignols, as advanced by Lu and Ralph (14) (Figure 6). It
is clear that tetrahydrofuran structures I and II can only be formed if sinapyl
alcohol is pre-acetylated (at monomer stage) and then undergoes EE coupling.
139
5. Resultados y discusin
t-Sac
100
relative intensity (a)
t-S
t-G
t-G ac
c-Sac
c-G acc-G c-S
0
t-Sac
100
t-S (b)
relative intensity
t-G
t-G
t-S
c-Sac
t-G ac c-S
c-G
0
100 t-Sac
t-S (d)
relative intensity
t-G
t-G ac
c-G c-Sac
c-G ac c-S
0
5.0 7.5 10.0 12.5 15.0
Retention time (min)
Figure 5. Chromatograms of the DFRC degradation products of MWL from (a) sisal, (b)
kenaf, (c) abaca and (d) curaua. c-G, t-G, c-S and t-S are the normal cis- and trans-guaiacyl
and syringyl monomers, respectively (as their propionylated derivatives). c-Gac, t-Gac, c-Sac
and t-Sac are the originally acetylated cis- and trans-guaiacyl and syringyl monomers,
respectively (as their propionylated derivatives).
140
5. Resultados y discusin
141
142
O O
=
=
CH 3O O
=
O-C-CH 3 O-C-CH 3 O O
=
=
J J O-C-CH 3
J J
CH 3-C-O O-C-CH 3
E E O-C-CH 3
E E
=
D D PrO
EE D D DFRC O
CH 3O OCH 3
O CH 3O
+ homo-coupling
HO OH
CH 3O OCH 3 CH 3O OCH 3 CH 3O OCH 3
OH OH OCH 3 I OCH 3
OPr
OH
O CH 3O OCH 3 O
=
CH 3O
=
CH 3O
OH O-C-CH 3 OPr O-C-CH 3
J J
O-C-CH 3 OPr
=
E E J
D D E
PrO O PrO
D
EE HO DFRC
+ O CH 3O + CH 3O
J
E D
cross-coupling O
=
CH 3-C-O
CH 3O OCH 3 CH 3O OCH 3 CH 3O OCH 3 CH 3O OCH 3
OCH 3
OH OH
OPr OPr
CH 3O OH
IIa; M += 630; [M-56]+= 574 IIb; M += 630: [M-56]+= 574
II
OCH 3
OH
OH OH CH 3O
J J O
J D OCH 3 OPr
E E
D D E E OPr
EE D J DFRC PrO
+ CH 3O O
homo-coupling CH 3O
HO III
CH 3O OCH 3 CH 3O OCH 3
OH OH OCH 3 CH 3O OCH 3
OPr
Figure 6. Structures of the tetrahydrofuran dimers arising from theEE coupling of sinapyl alcohol and sinapyl acetate. I:EE coupling product of
two sinapyl acetates; II: EE coupling product of a sinapyl alcohol and a sinapyl acetate; III: EEcoupling product (syringaresinol) of two sinapyl
alcohols. The aryltetralin products expected from the DFRC degradation of these tetrahydrofuran moieties are also shown, with indication of their
molecular weight and base peak in their mass spectra. Adapted from Lu and Ralph (14).
O
5. Resultados y discusin
IIa,IIb
100
I III
relative intensity
(a)
0
III
100
(b)
relative intensity
IIa,IIb
0
100
(c)
relative intensity
0
100
(d)
relative intensity
0
20.0 22.5 25.0 27.5 30.0 32.5
Retention time (min)
Figure 7. Detail of reconstructed chromatogram (sum of the characteristic ions at m/z 560,
574 and 580) of the DFRC degradation products of the MWL from (a) sisal, (b) kenaf, (c)
abaca and (d) curaua, showing the presence of aryltetralin EE products containing two (I),
one (IIa and IIb) and no (III) native acetates.
143
5. Resultados y discusin
144
5. Resultados y discusin
acylation, which in many plants takes place at very high levels, as seen above,
and which have often been overlooked in the past; otherwise the conclusions
drawn may not be representative of the real native lignin structure.
4. Conclusions
The structure of the MWL isolated from the herbaceous plants sisal, kenaf,
abaca and curaua has been elucidated by 2D-NMR and DFRC techniques. The
analyses indicated that the lignins from these plants are extensively acylated at
the J-carbon of the lignin side-chain (with either acetate and/or p-coumarate
groups) and preferentially on syringyl moieties. The structure of these highly
acetylated lignins can be essentially regarded as syringyl units linked mostly
through EO-4 ether bonds, where the J-carbons of the side-chains are
extensively acylated. The lignin polymer is therefore extremely linear and
unbranched. The study of highly acylated lignins will significantly contribute to
redefine the structure of lignin and to complete the lignin biosynthetic pathway.
Acknowledgements
This study has been supported by the Spanish MEC (projects AGL2005-01748
and BIO2007-28719-E) and the EU contract NMP2-CT-2006-26456
(BIORENEW). JR thanks the Spanish CSIC for an I3P fellowship; GM thanks
the Spanish Ministry of Education for a FPI fellowship.
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(Agave sisalana) plant materials at the gel state. Proc. EWLP-2008,
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Lore of the Plant Cell Wall Biosynthesis, Structure and Function, Hayashi,
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(43) Adler, E. Lignin chemistry past, present and future. Wood Sci. Technol.
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(44) Brunow, G. Methods to Reveal the Structure of Lignin. In: Hofrichter M &
Steinbchel A, (ed), Lignin, Humic Substances and Coal, Vol 1, 2001, pp.
89116, Wiley-VHC, Weinheim.
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Publicacin IV:
150
5. Resultados y discusin
Instituto de Recursos Naturales y Agrobiologa de Sevilla, CSIC, P.O. Box 1052, 41080-
Seville, Spain
Abstract
The chemical composition of leaf fibers of curaua (Ananas erectifolius), an
herbaceous plant native of Amazonia, was studied. Special attention was paid to
the content and composition of lignin and lipophilic compounds. The analysis of
lignin in the curaua fibers was performed in situ by pyrolysis-gas
chromatography/mass spectrometry (Py-GC/MS) and showed a lignin
composition with a p-hydroxyphenyl:guaiacyl:syringyl units (H:G:S) molar
proportion of 30:29:41 (S/G molar ratio of 1.4). The presence of p-
hydroxycinnamic acids (p-coumaric and ferulic acids) in curaua fibers was
revealed upon pyrolysis in the presence of tetramethylammonium hydroxide. On
the other hand, the main lipophilic compounds, analysed by GC/MS, were series
of long-chain n-fatty acids, n-fatty alcohols, D- and Z-hydroxyacids,
monoglycerides, sterols and waxes. Other compounds, such as Z-hydroxy
monoesters and Z-hydroxy acylesters of glycerol were also found in this fiber in
high amounts.
Keywords: Curaua; Ananas erectifolius; lipids; lignin; pyrolysis; hydroxy
monoesters; glyceryl esters; paper pulp.
1. Introduction
An alternative to wood raw materials for pulp and paper production in
developing countries is the use of non-woody fibers from herbaceous field
crops. In developed countries non-woody fibers are mainly used for the
production of specialty papers, i.e. tea bags, filter papers, bank notes, etc. The
main sources of non-woody raw materials are agricultural residues from
monocotyledons, including cereal straw and bagasse. Bamboo, reeds and some
other grass plants such as flax, hemp, kenaf, jute, sisal or abaca are also grown
or collected for the pulp industry but increased attention has been paid in recent
years to find new non-wood raw materials for pulp production.
Curaua (Ananas erectifolius), an herbaceous plant native of the Amazonian
region and member of the bromeliad family, has been recognized since pre-
Columbian days for its valuable fibers. In the last decade, it has gained
commercial recognition as material for composites for automotive industry [1-
4]. The curaua fiber has also been promoted for paper pulp in Brazil [5] and it is
151
5. Resultados y discusin
152
5. Resultados y discusin
content, the acetone extracted samples were subsequently extracted with hot
water (3 h at 100 C) to remove the water-soluble material. Holocellulose was
isolated from the pre-extracted fibers by delignification for 4 hours using the
acid chlorite method (19). The D-cellulose content was determined by removing
the hemicelluloses from the holocellulose by alkali extraction (19). Klason
lignin was estimated as the residue after sulfuric acid hydrolysis of the pre-
extracted material according to Tappi rule T222 om-88 [20]. The acid-soluble
lignin was determined, after filtering off the insoluble lignin, by
spectrophotometric determination at 205 nm wavelength. Neutral sugars from
polysaccharide hydrolysis were analyzed as alditol acetates by GC according to
Tappi rule T249 om85 [20]. Ash content was estimated as the residue after 6 h at
575 C. The general composition (as percent of whole fiber) was as follows:
holocellulose, 92.5%; D-cellulose, 66.4%; ash, 1.3%; acetone extractives, 5.3%;
water-soluble extract, 5.1%; Klason lignin, 4.9%; acid-soluble lignin, 1.6%. The
composition of neutral monosaccharides (as percent of total neutral
carbohydrates) included arabinose, 2.7%; xylose, 8.0%; mannose, 3.5%;
galactose, 0.2%; and glucose, 85.6%. No uronic acid determination was
performed in this study. The composition of metals and other elements was
analyzed by inductively coupled plasma spectrophotometry (ICP-OES) after
oxidation with concentrated HNO3 under pressure in a microwave digestor, with
the following results: K, 2770 ppm; Ca, 2025 ppm; Mg, 945 ppm; Mn, 120 ppm;
Na, 95 ppm; Al, 86 ppm; Fe, 82 ppm; Sr, 10 ppm; Zn, 4 ppm.
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5. Resultados y discusin
as the carrier gas at a rate of 5 mL/min, and the injection was performed in
splitless mode. Peaks were quantified by area, and a mixture of standards
(octadecane, palmitic acid, sitosterol and cholesteryl oleate) was used to
elaborate calibration curves. The data from the two replicates were averaged. In
all cases the standard derivations from replicates were below 10% of the mean
values.
The GC/MS analyses were performed with a Varian model Star 3400 GC
equipped with a model Saturn 2000 ion trap detector using a medium-length (12
m) capillary column of the same characteristics described above. The oven was
heated from 120 C (1 min) to 380 C at 10 C/min and held for 5 min. The
transfer line was kept at 300 C. The injector was temperature programmed from
120 C (0.1 min) to 380 C at a rate of 200 C/min and held until the end of the
analysis. Helium was used as the carrier gas at a rate of 2 mL/min. Methylation
with trimethylsilyldiazomethane and silylation with
bis(trimethylsilyl)trifluoroacetamide (BSTFA) was used when required.
Compounds were identified by comparing their mass spectra with mass spectra
in Wiley and NIST libraries, by mass fragmentography, and when possible, by
comparison with authentic standards.
2.4. Py-GC/MS
The pyrolysis of curaua fibers (approximately 100 Pg) was performed in
duplicate with a model 2020 micro-furnace pyrolyzer (Frontier Laboratories
Ltd., Yoriyama, Japan) directly connected to an Agilent 6890 GC/MS system
equipped with a 30 m 0.25 mm i.d., 0.25 Pm HP 5MS fused silica capillary
column. The detector consisted of an Agilent 5973 mass selective detector (EI at
70 eV). The pyrolysis was performed at 500 C. The final temperature was
achieved at a rate of 20 C/min. The GC/MS conditions were as follows: oven
temperature was held at 50 C for 1 min and then increased up to 100 C at
30 C/min, from 100 to 300 C at 10 C/min and isothermal at 300 C for
10 min. The carrier gas used was helium with a controlled flow of 1 ml/min. For
the pyrolysis in the presence of TMAH, approximately 100 Pg of sample was
mixed with 0.5 PL of 25% TMAH. The pyrolysis was carried out as described
above. The compounds were identified by comparing the mass spectra obtained
with those of the Wiley and NIST computer libraries and that reported in the
literature [16, 17]. Relative peak molar areas were calculated for carbohydrate
and lignin pyrolysis products. The summed molar areas of the relevant peaks
were normalized to 100%, and the data for two repetitive pyrolysis experiments
were averaged. The relative standard deviation for the pyrolysis data was less
than 5%.
154
5. Resultados y discusin
155
5. Resultados y discusin
11,12
6
19
10
5
3 18
2
15 23
34
24 32,33
13 16
14 20
7
40
17 21 37
22 27 29
25
38
26 28 30 31 35 36 39 42 43
41 44
2 4 6 8 10 12 14 16 18 20 22
Retention time (minutes)
Figure 1. Py-GC/MS chromatogram of curaua fibers. The identities and relative molar
abundances of the compounds are listed in Table 1.
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5. Resultados y discusin
Table 1. Identification and Relative Molar Abundances (%) of the Compounds Released after
Py-GC/MS of Curaua Fibers.
No Compound Mass Fragments MW Origin %
1 acetic acid 45/60 60 C 35.8
2 2-hydroxypropanal 43/74 74 C 3.1
3 (3H)-furan-2-one 55/84 84 C 3.1
4 1,3-hydroxydihydro-2-(3H)-furanone 58/102 102 C 6.0
5 (2H)-furan-3-one 55/84 84 C 1.4
6 2-furaldehyde 67/95/96 96 C 6.4
7 cyclopent-1-ene-3,4-dione 54/68/96 96 C 0.9
8 (5H)-furan-2-one 55/84 84 C 4.2
9 2,3-dihydro-5-methylfuran-2-one 55/69/98 98 C 8.5
10 4-hydroxy-5,6-dihydro-(2H)-piran-2-one 58/85/114 114 C 2.1
11 3-hydroxy-2-methyl-2-cyclopenten-1-one 55/85/112 112 C 1.1
12 2-hydroxy-3-methyl-2-cyclopenten-1-one 55/85/112 112 C 4.8
13 4-methylphenol 77/107/108 108 LH 0.7
14 guaiacol 81/109/124 124 LG 0.5
15 2 furoic acid, methyl ester 67/95/126 126 C 1.3
16 4-methylguaiacol 95/123/138 138 LG 0.1
17 3,4-dihydroxybenzaldehyde 81/109/137/138 138 L 0.5
18 catechol 64/81/92/110 110 L/C 1.0
19 4-vinylphenol 65/91/120 120 LH/pCA 2.2
20 5-hydroxymethyl-2-furaldehyde 69/97/109/126 126 C 1.9
21 3-methoxycatechol 60/97/125/140 140 L 0.3
22 4-ethylguaiacol 122/137/152 152 LG 0.2
23 4-vinylguaiacol 107/135/150 150 LG 1.1
24 syringol 111/139/154 154 LS 0.7
25 eugenol 131/149/164 164 LG 0.2
26 4-propylguaiacol 122/136/166 166 LG <0.1
27 vanillin 109/151/152 152 LG 0.3
28 cis-isoeugenol 131/149/164 164 LG <0.1
29 4-methylsyringol 125/153/168 168 LS 0.2
30 trans-isoeugenol 131/149/164 164 LG 0.1
31 acetoguaiacone 123/151/166 166 LG 0.1
32 levoglucosane 60/98 162 C 7.7
33 4-ethylsyringol 167/182 182 LS 0.8
34 4-vinylsyringol 137/165/180 180 LS 0.9
35 4-allylsyringol 167/179/194 194 LS 0.1
36 4-propylsyringol 123/167/196 196 LS 0.1
37 cis-4-propenylsyringol 167/179/194 194 LS 0.2
38 syringaldehyde 167/181/182 182 LS 0.2
39 4-propinylsyringol 106/131/177/192 192 LS 0.1
40 trans-4-propenylsyringol 167/179/194 194 LS 0.5
41 trans-coniferaldehyde 107/135/147/178 178 LG 0.1
42 acetosyringone 153/181/196 196 LS 0.1
43 syringylacetone 123/167/210 210 LS 0.1
44 trans-sinapaldehyde 137/165/180/208 208 LS 0.1
%H 29.8
%G 29.1
%S 41.1
S/G 1.4
%L 11.7
%C 88.3
L/C 0.13
C, carbohydrates; L, lignin; LH, p-hydroxyphenyl lignin units, H; LG, guaiacyl lignin units, G; LS, syringyl
lignin units, S; pCA, p-coumaric acid.
157
5. Resultados y discusin
OHM24
3,4
OH20 OH24
OHM26
Al22
Al24
M24
OH22
OH22
1
OH24
2
M26
W38
FA18:2 FA24 SG
FA16:1 + FA22
FA18:1
OHM28
FA18 FA20 M28
FA16 W40 CG
W42
CE SE
5 10 15 20 25
Retention time (minutes)
Figure 2. GC/MS chromatogram of the methyl ester and TMSi ether derivative of the lipid
extract from curaua fibers. FA(n), n-fatty acid series; Al(n), alcohol series; W(n), wax series;
DOH(n) and ZOH(n), D and Z-hydroxy fatty acids series; M(n), monoglyceride series;
ZOHM(n), Z-hydroxy acylesters of glycerol series; SG, sitosteryl 3E-D-glucopyranoside; CG,
campesteryl 3E-D-glucopyranoside;1, campesterol; 2, ergostanol; 3, sitosterol; 4,
stigmastanol; CE, campesterol ester; SE, sitosterol ester; n denotes the total carbon atom
number.
158
5. Resultados y discusin
sterols 618.7
campesterol 55/145/213/382/400 400 56.9
ergostanol 215/402 402 145.7
sitosterol 145/213/396/414 414 226.4
stigmastanol 215/416 416 189.7
tocopherols 31.4
D-tocopherol 165/205/430 430 31.4
159
5. Resultados y discusin
waxes 173.2
C36 201/229/257/285/536 536 3.5
C37 243/257/550 550 3.3
C38 257/564 564 56.8
C39 243/257/271/285/299/578 578 5.9
C40 257/285/313/592 592 46.5
C40:1 264/283/590 590 2.9
C41 257/271/285/299/313/327/341/355/606 606 2.3
C42 257/285/313/341/620 620 24.9
C42:1 264/283/618 618 1.2
C43 257/271/285/299/313/327/355/369/634 634 1.5
C44 257/285/313/341/648 648 19.1
C46 257/285/313/341/369/397/676 676 5.3
monoglycerides 714.6
1-monotetradecanoylglycerol 73/103/129/147/343/431* 446* 5.1
1-monohexadecanoylglycerol 73/103/129/147/371/459* 474* 5.3
1-monooctadecanoylglycerol 73/103/129/147/399/487* 502* 5.3
1-monoeicosanoylglycerol 73/103/129/147/427/515* 530* 31.6
1-monodocosanoylglycerol 73/103/129/147/455/543* 558* 288.2
1-monotetracosanoylglycerol 73/103/129/147/483/571* 586* 179.3
1-monohexacosanoylglycerol 73/103/129/147/511/599* 614* 168.2
160
5. Resultados y discusin
Waxes (esters of fatty acids to fatty alcohols) were also important components
of the curaua fiber extracts and were found in the range from C36 to C46. Among
the waxes, the GC/MS analysis revealed that each chromatographic peak
consisted of a complex mixture of different long-chain fatty acids esterified to
different long-chain fatty alcohols. The identification and quantification of the
individual long-chain esters in each chromatographic peak was resolved based
on the mass spectra of the peaks. The mass spectra of long-chain esters are
characterized by a base peak produced by a rearrangement process involving the
transfer of 2H atoms from the alcohol chain to the acid chain giving a protonated
acid ion [24, 43-45]. Therefore, the base peak gives the number of carbon atoms
in the acid moiety and the molecular ion the total number of carbon atoms in the
ester. It is possible then to determine the individual contribution of the esters to
every chromatographic peak by mass spectrometric determination of the
molecular ion and the base peak. Quantification of individual esters was
accomplished by integrating areas in the chromatographic profiles of ions
characteristic for the acidic moiety. The detailed structural composition and
abundance of the high molecular weight waxes identified in the curaua fiber is
shown in Table 3. The esterified fatty acids ranged from C12 to C25 and the
esterified fatty alcohols ranged from C16 to C30. Waxes with unsaturated fatty
acids (C40:1 and C42:1) were also found in lower amounts, the unsaturated fatty
acid being in all cases oleic acid.
161
5. Resultados y discusin
162
5. Resultados y discusin
C18:1:C24 1.2
163
5. Resultados y discusin
Z-Hydroxy fatty acids esterified to glycerol were also found in high amounts
in the curaua fiber. The mass spectra of the TMSi derivatives of Z-hydroxy
acylesters of glycerol are characterized by the presence of an abundant fragment
arising from the loss of a methyl group at [M-15]+. The cleavage between the C-
2 and C-3 carbons in the glyceryl moiety gives rise to the fragments at m/z 103
and [M-103]+. Other diagnostic ions are derived from the glyceryl moiety- i.e. at
m/z 205 as a result of the cleavage between the C-2 and C-1 (the esterified
carbon), and at m/z 219 due to the loss of the acyloxy moiety. The same loss of
the acyloxy group from M+ and M-15+, but with the H rearrangement, gives rise
to the ions at m/z 218 and 203, respectively. Other significant ions in the low-
mass region occur at m/z 73 (the TMSi group), m/z 129 (the glycerol carbon
backbone with a TMSi group [H2C=CHCH=O+Si(CH3)3]) and m/z 147
(produced by the rearrangement of two TMSi groups) [51]. The Z-hydroxy fatty
acids esterified to the glycerol ranges from C22 to C28. The structure and mass
spectrum of the TMSi derivative of 1-mono-(22-hydroxydocosanoyl)glycerol is
shown in Figure 5.
164
5. Resultados y discusin
OH
A
OH
B
O
HO
OH
C
O
OH
D OH
O
HO
O
E
O-CH2
HO-O-CH
F
HO-O-CH2
O
HO
O-CH2
HO-O-CH
G
HO-O-CH2
HO HO HO HO
H I J K
CH2OH CH2OH
O O
OH O OH O
HO HO
OH L OH M
Figure 3. Structures of the main lipids present in the curaua fibers. A: stearic acid, B: n-
docosanol, C: 26-hydroxyhexacosanoic acid, D: 2-hydroxytetracosanoic acid, E: docosanyl,
16-hydroxyhexadecanoate, F: 1-monodocosanoylglycerol, G: 1-mono(24-
hydroxytetracosanoyl)glycerol, H: campesterol, I: ergostanol, J: sitosterol, K: stigmastanol, L:
campesteryl 3E-D-glucopyranoside, M: sitosteryl 3E-D-glucopyranoside.
165
5. Resultados y discusin
[M-15]+
[M-15-C22H45OH] +
637
100% 311
n-Fatty alcohols ranging from C20 to C28 were present in the curaua extracts
with the presence of only the even carbon atom homologs, docosanol (C22) and
tetracosanol (C24) being the most abundant. Monoglycerides, accounting for
690.7 mg/Kg of the fibers, were present in important amounts, from C14 to C30,
C22 (1-monodocosanoylglycerol) being the most prominent. Di- and
triglycerides were only identified in trace amounts.
Sterols were also present among the lipids of curaua fibers in high amounts.
Sitosterol was the most abundant among the free sterols with the presence of
minor amounts of stigmastanol, ergostanol and campesterol. Lower amounts of
sitosterol and campesterol could also be found in ester form. Sterol glycosides,
such as sitosteryl and campesteryl 3E-D-glucopyranosides were also identified
in high amounts, the former being the most predominant. The identification of
steryl glycosides was accomplished, after BSTFA derivatization of the lipid
extract, by comparison with the mass spectra and relative retention times of
authentic standards [52]. Finally, other compounds identified among the curaua
fiber extractives were D-tocopherol, several steroid hydrocarbons and steroid
ketones, as reflected in Table 2.
In conclusion, curaua fiber is characterized by a high content of holocellulose
and D-cellulose and low lignin content which would make this fiber suitable for
papermaking. Moreover, the lignin composition indicates a slight predominance
of S-lignin units (S/G molar ratio of 1.4). On the other hand, the high extractive
content can be considered as a detrimental aspect, however most of the acetone
extracts are due to polar compounds and only 1.3% corresponds to lipophilic
compounds. Indeed, most of the lipophilic compounds are easily saponifiable,
and therefore can be hydrolyzed and dissolved during alkaline cooking.
166
5. Resultados y discusin
73
OTMS
100%
OTMS
O O
TMS O
129
103 147
203
[M-15]+
321 631
[M-145]+ [M-88]+
411 486
381 543
265
Acknowledgements
This study has been supported by the Spanish MEC (project AGL2005-01748).
We thank CELESA (Tortosa, Spain) for providing the curaua fibers.
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Mark, S.H. Kandil, and Z.H. Kafafi, eds) Science and Technology of Polymers
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spectrometry. J. Anal. Appl. Pyrol. 1999, 49, 295-305.
[7] del Ro, J. C.; Gutirrez, A.; Hernando, M.; Landn, P.; Romero, J.;
Martnez, A. T. Determining the influence of eucalypt lignin composition in
paper pulp yield using Py-GC/MS. J. Anal. Appl. Pyrol. 2005, 74, 110-115.
[8] Nimz, H. Beech lignin- Proposal of a constitutional scheme. Angew. Chem.
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[9] Adler, E. Lignin chemistry - Past, present and future. Wood Sci. Technol.
1977, 11, 169-218.
[10] Tsutsumi, Y.; Kondo, R.; Sakai, K.; Imamura, H. The difference of
reactivity between syringyl lignin and guaiacyl lignin in alkaline systems.
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[11] Hillis, W. E.; Sumimoto, M. Effect of extractives on pulping. In Natural
Products of Woody Plants II; Rowe, J. W., Ed.; Springer-Verlag, Berlin,
Germany, 1989; pp 880-920.
[12] Back, E. L.; Allen, L. H. Pitch Control, Wood Resin and Deresination;
Tappi Press: Atlanta, GA, 2000.
[13] Gutirrez, A.; del Ro, J. C.; Martnez, M. J.; Martnez, A. T. The
biotechnological control of pitch in paper pulp manufacturing. Trends
Biotechnol. 2001, 19, 340-348.
[14] Ali, M.; Sreekrishnan, T. R. Aquatic toxicity from pulp and paper mill
effluents: A review. Adv. Environ. Res. 2001, 5, 175-196.
[15] Rigol, A.; La Torre, A.; Lacorte, S.; Barcel, D. Bioluminiscence
inhibition assays for toxicity screening of wood extractives and biocides in
paper mill process waters. Environ. Toxicol. Chem. 2003, 23, 339-347.
[16] Faix, O.; Meier, D.; Fortmann, I. Thermal degradation products of wood. A
collection of electron of electron-impact (EI) mass spectra of monomeric lignin
derived products. Holz Roh- Werkst. 1990, 48, 351-354.
[17] Ralph, J.; Hatfield, R. D. Pyrolysis-GC/MS characterization of forage
materials. J. Agric. Food Chem. 1991, 39, 1426-1437.
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[18] Gutirrez, A.; del Ro, J. C.; Gonzlez-Vila, F. J.; Martn, F. Analysis of
lipophilic extractives from wood and pitch deposits by solid-phase extraction
and gas chromatography. J. Chromatogr. A. 1998, 823, 449-455.
[19] Browning, B.L. Methods of Wood Chemistry, vol. II; Wiley-Interscience
Publishers, New York, USA, 1967.
[20] Technical Association of the Pulp and Paper Industry. Test methods, 1992-
1993. TAPPI, Atlanta, GA. 1993.
[21] Gutirrez, A.; Rodrguez, I. M.; del Ro, J. C. Chemical characterization of
lignin and lipid fractions in kenaf bast fibers used for manufacturing high-
quality papers. J. Agric. Food Chem. 2004, 52, 4764-4773.
[22] van Dam, J. E. G.; van Vilsteren, G. E. T.; Zomers, F. H. A.; Shannon, W.
B.; Hamilton, I. T. Increased application of domestically produced plant fibers
in textiles, pulp and paper production and composite materials. Directorate-
General XII, Science, Research and Development, European Commision:
Brussels, 1994.
[23] del Ro, J. C.; Gutirrez, A. Chemical composition of abaca (Musa textilis)
leaf fibers used for manufacturing of high quality paper pulps. J. Agric. Food
Chem. 2006, 54, 4600-4610.
[24] Gutirrez. A.; del Ro, J. C. Lipids from flax fibers and their fate in alkaline
pulping. J. Agric. Food Chem. 2003, 51, 4965-4971.
[25] Gutirrez. A.; del Ro, J. C. Lipids from flax fibers and their fate in alkaline
pulping. J. Agric. Food Chem. 2003, 51, 6911-6914.
[26] Gutirrez, A.; Rodrguez, I. M.; del Ro, J. C. Chemical characterization of
lignin and lipid fractions in industrial hemp bast fibers used for manufacturing
high-quality paper pulps. J. Agric. Food Chem. 2006, 54, 2138-2144.
[27] Moore, G. Nonwood Fibre Applications in Papermaking; Pira International,
Leatherhead, Surrey, UK, 1996
[28] del Ro, J. C.; Martn, F.; Gonzlez-Vila, F. J. Thermally assisted
hydrolysis and alkylation as a novel pyrolytic approach for the structural
characterization of natural biopolymers and geomacromolecules. Trends Anal.
Chem. 1996, 15, 70-79.
[29] Lam, T. B. T.; Kadoya, K.; Liyama, K. Bonding of hydroxycinamic acids
to lignin: ferulic and p-coumaric acids are predominantly linked at the benzyl
position of lignin, not the E-position, in grass cell walls. Phytochemistry 2001,
57, 987-992.
169
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170
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171
5. Resultados y discusin
Publicacin V:
Coelho D., Marques G., Gutirrez A., Silvestre A.R.D. and del Ro J.C. (2007)
Chemical characterization of the lipophilic fraction of Giant reed (Arundo donax)
fibers used for pulp and paper manufacturing. Industrial Crops and Products, 26,
229-236.
172
5. Resultados y discusin
Abstract
The chemical composition of lipophilic extractives from Arundo donax stems
(including nodes and internodes), used for pulp and papermaking, was studied.
The lipid fraction was extracted with acetone and redissolved in chloroform, and
then fractionated by solid-phase extraction (SPE) on aminopropyl-phase
cartridges into four different fractions of increasing polarity. The total lipid
extract and the resulting fractions were analysed by gas chromatography and gas
chromatography-mass spectrometry, using short- and medium-length high-
temperature capillary columns, respectively. The main compounds identified in
the fibres included series of long-chain n-fatty acids, n-alkanes, n-aldehydes, n-
alcohols, monoglycerides, free and esterified sterols and triterpenols, steryl
glucosides, steroid hydrocarbons and steroid and triterpenoid ketones. Minor
amounts of other compounds such as diglycerides, waxes and tocopherols were
also identified among the lipids of A. donax.
Keywords: Arundo donax, lipophilic extractives, pitch, fatty acids, sterols, steryl
glucosides, GC, GC/MS.
1. Introduction
In the last decades, fast growing plants have received particular attention as
alternative sources of cellulose fibres (van Dam et al. 1994; Moore, 1996).
These non-wood plants are the common fibre source for paper pulp production
in developing countries where wood fibres are not available. In the developed
world, although wood is still by far the main raw material for pulp and paper
manufacture, a market exists for high-value-added papers from these fibres.
Arundo donax L. (giant reed) is a widely distributed naturally growing perennial
rhizomatous grass with a segmented tubular structure like bamboo (Seca et al.,
2000), which has been considered as one of the promising non-wood plants for
pulp and paper industry (Shatalov and Pereira, 2002). The easy adaptability to
different ecological conditions, the annual harvesting period and the high
biomass productivity (32-37 t per year-1ha-1 of dry biomass) reached by intensive
cultivation (Vecchiet et al., 1996), combined with appropriate chemical
composition (Shatalov et al., 2001), make A. donax very attractive as an
alternative source of fibres (Shatalov and Pereira, 2005).
173
5. Resultados y discusin
2. Experimental
2.1. Samples
Samples of A. donax L. reed stems (including nodes and internodes) were
supplied by University of Huelva, Spain. The samples were air-dried and milled
using a knife mill (Janke and Kunkel, Analysenmhle). For the isolation of
174
5. Resultados y discusin
lipids, the milled samples were Soxhlet extracted with acetone for 8h. The
lipophilic extractives were obtained by redissolving the dried acetone extract in
chloroform and evaporated to dryness under nitrogen.
175
5. Resultados y discusin
mass spectra in Wiley and NIST libraries, by mass fragmentography, and, when
possible, by comparison with authentic standards.
Sitosterol
FA28
SG
Campesterol
FA30
FA18:1
Stigmasterol
FA18:2
CG
FA26
StG
FA24
FA17 FA18 MG26
MG16 MG18 Steryl/triterpenyl esters
FA20
5 10 15 20 30
Retention time (min)
Figure 1. GC/MS chromatogram of the derivatized (TMS) chloroform extract of Arundo
donax fibres. FA: fatty acids; MG: monoglycerides; CG: campesteryl 3E-D-glucopyranoside;
StG: stigmasteryl 3E-D-glucopyranoside; SG: sitosteryl 3E-D-glucopyranoside.
176
5. Resultados y discusin
Ak 29 A
Steryl esters
Ak 27
Ak 25 Ak 31
5 10 15 20 25
Cycloartenone
B
-amyrenone
-amyrenone
5 10 15 20 25
-Sitosterol + Stigmastanol + -amyrin
C
Campesterol
Stigmasterol -amyrin
7-oxositosterol
5 10 15 20 25
FA28 D
FA26
FA16
FA18:1
+
FA18:2 FA24
FA18
5 10 15 20 25
Figure 2. GC/MS chromatograms of the different SPE fractions isolated from the A. donax
fibres extracts. Fraction A, eluted with 8 mL of hexane; fraction B, eluted with 6mL of
hexane:chloroform (5:1); fraction C, eluted with 10 mL of chloroform; and fraction D, eluted
with 10 mL diethyl ether:acetic acid (98:2). FA: fatty acids; AK: n-alkanes.
177
5. Resultados y discusin
178
5. Resultados y discusin
Aldehydes 81.6
n-hexacosanal 82/96/362 380 10.4
n-octacosanal 82/96/390 408 22.9
n-triacontanal 82/96/418 436 48.3
Sterols/Triterpenols 528.1
campesterol 55/145/213/382/400 400 90.6
stigmasterol 55/81/255/394/412 412 46.4
sitosterol 145/213/396/414 414 281.0
stigmastanol 215/416 416 71.9
7-oxo-sitosterol 135/161/187/396/428 428 6.5
-amyrin 189/203/218/409/426 426 8.2
-amyrin 189/203/218/409/426 426 23.5
Tocopherol 17.7
-tocopherol 151/416 416 6.8
-tocopherol 165/430 430 10.9
Monoglyceride 367.5
2,3-dihydroxypropyl tetradecanoate 73/103/129/147/343/431 * 446 * 5.5
2,3-dihydroxypropyl hexadecanoate 73/103/129/147/371/459 * 474* 94.2
2,3-dihydroxypropyl octadecanoate 73/103/129/147/399/487 * 502 * 86.6
2,3-dihydroxypropyl eicosanoate 73/103/129/147/427/515 * 530 * 35.1
2,3-dihydroxypropyl docosanoate 73/103/129/147/455/543 * 558 * 43.0
2,3-dihydroxypropyl tetracosanoate 73/103/129/147/483/571 * 586 * 46.9
2,3-dihydroxypropyl hexacosanoate 73/103/129/147/511/599 * 614 * 56.2
179
5. Resultados y discusin
Diglycerides 47.6
dipalmitin, 1,2- (P2) 57/129/313/386/625 * 640 * 7.8
dipalmitin, 1,3- (P2) 57/129/314/371/385/625 * 640 * 12.1
palmitoylstearin (PS) 57/129/314/372/399/579 * 668 * 16.8
distearin, 1,2- and 1,3- (S2) 57/129/342/399/607 * 696 * 10.9
* as TMSi ether derivates; bold mass fragments indicate base peaks.
enriched in free fatty acids was eluted with diethyl ether-acetic acid (98:2). The
identities and abundances of the main compounds identified are listed in Table
1. The most predominant lipid classes identified among the A. donax lipid
extracts were series of n-fatty acids (41% of total lipids identified), sterols
(19%), monoglycerides (13%), fatty alcohols (7%) and steryl glucosides (6%).
Minor amounts of alkanes, aldehydes, tocopherols, steroid hydrocarbons, steroid
and triterpenoid ketones and steryl/triterpenyl esters, were also present in these
fibres. The structures of main and representative compounds are shown in
Figure 3.
The series of free fatty acids were identified in A. donax fibres ranging from
tetradecanoic (C14) to dotriacontanoic (C32) acids, with strong even-over-odd
carbon atom predominance. Hexadecanoic acid (palmitic acid, I) was the most
abundant fatty acid, however a bimodal distribution, with a second maximum
for octacosanoic acid (C28) was observed. The unsaturated 9-octadecenoic
(oleic acid, II) and 9,12-octadecadienoic (linoleic acid, III) acids were also
present in important amounts. The series of n-alkanes was also identified in the
A. donax fibre ranging from docosane (C22) to tritriacontane (C33), with a
strong odd-over-even carbon atom number predominance, and nonacosane (IV)
being the most predominant homolog. n-Fatty alcohols ranging from
hexacosanol (C26) to dotriacontanol (C32) were present in the A. donax extracts
with the presence of only the even carbon atom number homologues,
triacontanol (V) being the most abundant. Significant amounts of a series of n-
aldehydes ranging from hexacosanal (C26) to triacontanal (C30) were identified
in the A. donax fibres with triacontanal (VI) predominating. Monoglycerides
were also present in high amounts in A. donax fibres. The series of
monoglycerides was identified in the range from C14 to C26, with maximum for
monopalmitin, C16, (VII).
Steroids and triterpenoids, including free sterols, steryl esters, steryl glucosides,
steroid ketones and hydrocarbons are among the most predominant compounds
in the lipophilic extract of A. donax fibre. Free sterols were the major compound
class among steroids and triterpenoids, sitosterol (VIII) being the main sterol
present. Other sterols, such as campesterol (IX), stigmasterol (X), stigmastanol
(XI) and the oxidized 7-oxositosterol, were also present. Steryl esters were also
present in A. donax extract, although in low amounts. The complete
identification of the individual steryl esters by GC-MS was not possible since
they only show fragments arising from the sterol moiety by electro-impact MS
180
5. Resultados y discusin
Figure 3. Structures of the main lipophilic compounds present in A. donax fibres. (I) palmitic
acid, (II) oleic acid, (III) linoleic acid, (IV) nonacosane, (V) triacontanol, (VI) triacontanal,
(VII) monopalmitin, (VIII) sitosterol, (IX) campesterol, (X) stigmasterol, (XI) stigmastanol,
(XII) sitosteryl 3E-D-glucopyranoside, (XIII) D-amyrin, (XIV) E-amyrin, (XV) stigmasta-3,5-
diene, (XVI) stigmasta-3,5,7-triene, (XVII) E-amyrenone, (XVIII) D-amyrenone, (XIX)
cycloartenone, (XX) stigmasta-3,5-dien-7-one, (XXI) stigmast-4-en-3-one, (XXII) stigmasta-
3,6-dione.
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5. Resultados y discusin
and rarely give detectable molecular ions (Lusby et al. 1984, Evershed et al.
1989). By monitoring the ions corresponding to the different sterol moieties in
the SPE fraction enriched in steryl esters, it was possible to identify series of
sitosterol as well as D- and E-amyrin esters. Steryl glucosides, such as
campesteryl, stigmasteryl and sitosteryl -D-glucopyranosides (XII), were
identified in significant amounts, the latter being the most predominant.
The identification of steryl glucosides was accomplished (after BSTFA
derivatization of the lipid extract) by comparison with the mass spectra and
relative retention times of authentic standards (Gutirrez and del Ro, 2001).
Among triterpenols, D-amyrin (XIII) and E-amyrin (XIV) occurred in free and
esterified form, with the latest being detected in low amounts. Finally, several
steroid hydrocarbons, such as stigmasta-3,5-diene (XV) and stigmasta-3,5,7-
triene (XVI) and triterpenoid and steroid ketones, such as E-amyrenone (XVII),
D-amyrenone (XVIII), cycloartenone (XIX), stigmasta-3,5-dien-7-one (XX),
stigmast-4-en-3-one (XXI) and stigmasta-3,6-dione (XXII), were also identified.
The different lipid classes present in A. donax fibres will have different
behavior during pulping and bleaching and therefore the problematic of pitch
will be different depending the type of pulping (i.e. mechanical, chemical) and
bleaching (ECF, TCF) processes. The knowledge of the chemical composition
of the lipophilic components of A. donax fibres shown here will assist to predict
pitch problems during pulp and papermaking of this fibre and to establish
appropriate methods for their control.
Acknowledgements
This study has been funded by the Spanish project AGL2005-01748. GM thanks
the Spanish Ministry of Education and Science for a FPI fellowship. We thank
M.J. Diaz (University of Huelva) for the Arundo donax fibres.
References
del Ro, J.C., Gutirrez A., Gonz.lez-Vila F.C., Martn F. and Romero J., 1998.
Characterization of organic deposits produced in kraft pulping of Eucalyptus
globulus wood. J. Chromatogr. A. 823, 457-465.
del Ro, J.C.; Romero, J.; Gutirrez, A., 2000. Analysis of pitch deposits
produced in Kraft pulp mills using a totally chlorine free bleaching sequence.
J. Chromatogr. A 874, 235-245.
Driss, M., Rozmarin, G. and Chene, M., 1973. Some physicochemical properties
of two xylans of reed (Phragmites communis and Arundo donax) in solution.
Cell. Chem. Technol. 7, 703-713.
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Evershed, R.P., M.C. Prescott, N. Spooner and L.J. Goad. 1989. Negative ion
ammonia chemical ionization and electron impact ionization mass
spectrometric analysis of steryl fatty acyl esters. Steroids 53, 285309.
Faix, O., Meier, D., Beinhoff, O., 1989. Analysis of lignocelluloses and lignins
from Arundo donax and Miscanthus sinensis Anderss and hydroliquefaction
of Miscanthus. Biomass 18, 109.
Freire, C.S.R., Silvestre, A.J.D. and Pascoal Neto, C., 2005. Lipophilic
extractives in Eucalyptus globulus Kraft pulps. Behaviour during ECF
bleaching. J. Wood Chem. Technol. 25, 67-80.
Gutirrez, A., del Ro, J.C., Gonzlez-Vila F.J. and Martn F., 1998. Analysis of
lipophilic extractives from wood and pitch deposits by solid-phase extraction
and gas chromatography. J. Chromatogr. A. 823, 449-455.
Gutirrez, A.; del Ro, J. C., 2003. Lipids from flax fibers and their fate in
alkaline pulping. J. Agric. Food Chem., 51, 4965-4971.
Gutirrez, A., del Ro, J.C. and Martnez, A.T., 2004. Chemical Analysis and
Biological Removal of Wood Lipids forming Pitch deposits in paper pulp
manufacturing. In: F.J.T. Spencer and A.L. Ragout de Spencer (Eds.),
Protocols in Environmental Microbiology. In: Methods in Molecular Biology.
Chapter 19, Humana Press, 2004, pp. 189-202.
Joseleau, J.P. and Barnoud, F., 1976. Cell wall carbohydrates and structural
studies of xylan in relation to growth in the Arundo donax. Appl. Polym.
Symp. 28, 983-992.
Joseleau, J.P., Miksche, G.E. and Yasuda, S., 1976. Structural variation of
Arundo donax lignin in relation to growth. Holzforschung 31, 19-20.
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Pascoal Neto, C., Seca, A., Nunes, A.M., Coimbra, M.A., Domingues, F.,
Evtuguin, D., Silvestre, A.J.D, Cavaleiro, J.A.S., 1997. Variations in chemical
composition and structure of macromolecular components in different
morphological regions and maturity stages of Arundo donax. Ind. Crops Prod.
6, 51-58.
Rigol, A.; La Torre, A.; Lacorte, S.; Barcel, D., 2003. Bioluminiscence
inhibition assays for toxicity screening of wood extractives and biocides in
paper mill process waters. Environ. Toxicol. Chem., 23, 339-347.
Shatalov, A.A., Quilh, T., Pereira, H., 2001. Arundo donax L. reed new
perspectives for pulping and bleaching. 2. Raw material characterization.
TAPPI J. 84 (1), 1-12.
Shatalov, A.A., Pereira, H., 2002. Influence of stem morphology on pulp and
paper properties of Arundo donax L. reed. Ind. Crops Prod. 15, 77-83.
Tai, D., Cho, W. And Ji, W., 1987. Studies of Arundo donax lignins.
Proceedings of the 4th ISWPC, Vol. II, April 1987, Paris, pp. 13-17.
van Dam, J. E. G.; van Vilsteren, G. E. T.; Zomers, F. H. A.; Shannon, W. B.;
Hamilton, I. T., 1994. Industrial fibre crops - study on: increased application
of domestically produced plant fibres in textiles, pulp and paper production
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Vecchiet, M., Jodice, R., Schenone, G., 1996. Agronomic research on giant reed
(Arundo donax L.). Management system and cultivation of two different
provenances. In: Chartier, Ph., Ferrero, G.L., Henius, U.M., Hultberg, S.,
Sachau, J., Wiinblab, M. (Eds.), Biomass for Energy and the Environment. In:
Proceedings of the Ninth European Biomass Conference, Copenhagen,
Pergamon, UK, pp. 644-648.
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Publicacin VI:
186
5. Resultados y discusin
Abstract
The chemical characterization of trimming residues of tagasaste (Chamaecytisus
proliferus spp. palmensis), a hardy leguminous shrub that has been recently
explored for pulp and paper production, was performed with especial emphasis
in the composition of lignin and lipophilic extractives. Tagasaste was
characterized by a high content of holocellulose (81%) and D-cellulose (41%),
while having a relatively low lignin content of 18.9%. The analysis of lignin was
performed in situ by pyrolysis-gas chromatography/mass spectrometry (Py-
GC/MS) and showed a composition with a guaiacyl:syringyl (G:S) molar
proportion of 38:62 (S/G molar ratio of 1.6) and the absence of p-hydroxyphenyl
(H) units. The high S/G ratio, together with its low lignin content makes
tagasaste an adequate raw material for pulping. On the other hand, the relatively
high acetone extractive content (1.4%) was mostly due to polar compounds and
only 0.2% corresponded to lipophilic compounds. The lipophilic compounds,
analyzed by GC and GC/MS, were mainly composed of fatty acids, including D-
hydroxyfatty acids, and steroid compounds, such as free and conjugated (esters
and glycosides) sterols.
Keywords: Tagasaste; lipids; lignin; pyrolysis; paper pulp.
1. Introduction
Tagasaste (Chamaecytisus proliferus spp. palmensis), also known as tree
lucerne, is a hardy leguminous and fast-growing shrub of the Fabaceae
(Genisteae) family. It is indigenous of the Canary Islands (Spain) but is now
being cultivated in Australia, New Zealand and other countries (Francisco-
Ortega et al., 1991). The shrub is being mainly exploited for high-protein fodder
to maintain livestock (Borens and Poppi, 1990; Ventura et al., 2002) and also as
N-fixing crops to improve soil fertility (Kindu et al., 2006). In order to
encourage the formation of bushes with multiple stems the shrub must be grazed
with regularity, which leads to a high accumulation of trimming residues. These
residues are nowadays considered as agricultural waste since they cannot be
converted to valuable products.
As attempts to reduce the adverse environmental impact and to use this
renewable biomass, they have been recently explored as an alternative raw
material for pulp production (Daz et al. 2004; Lpez et al., 2004; Jimnez et al.,
187
5. Resultados y discusin
2006; Jimnez et al., 2007; Garca et al., 2007). Tagasaste has been found to be
an excellent raw material for paper pulp production, similar to eucalypt wood,
with high hollocellulose and D-cellulose contents, and low lignin and extractives
content, giving high yields (Daz et al. 2004; Lpez et al., 2004; Jimnez et al.,
2006). However, despite all this previous work, a detailed chemical composition
of tagasaste trimming residues has not been addressed so far, which is of high
importance for optimizing the use of this raw material for paper pulp production.
The content and chemical structure of woody components, in particular the
lignin content and its composition in terms of its p-hydroxyphenyl (H), guaiacyl
(G) and syringyl (S) moieties are important parameters in pulp production in
view of delignification rates, chemical consumption and pulp yields. The higher
reactivity of the S lignin with respect to the G lignin in alkaline systems is
known (Chang and Sarkanen 1973; Tsutsumi et al. 1995) and therefore, the
lignin S/G ratio in hardwoods affects the pulping efficiency. It has already been
shown for eucalypt woods that higher S/G ratios imply higher delignification
rates, less alkali consumption and therefore higher pulp yield (Gonzlez-Vila et
al. 1999; del Ro et al. 2005).
On the other hand, the composition of extractives, especially the lipophilic
compounds, is also important for pulp and paper production. The different
classes of lipids have different behavior during cooking and bleaching. The
lipids can be classified into two principal groups, namely fatty acids and neutral
components, the latter including waxes, long chain n-fatty alcohols, alkanes and
steroids and triterpenoids. And the behavior of the fatty acids in an aqueous
environment is quite different from that of the neutrals. In alkaline pulping, the
acids dissociate and can dissolve in water to quite a high extent, forming fatty
acid soaps. The neutrals, however, have a very low solubility in water and
survive the cooking process and remain in the pulp being at the origin of the so-
called pitch deposits, which are responsible of reduced product quality and
higher operating costs due to production stops for cleaning the equipment (Hillis
and Sumimoto 1989; Back and Allen 2000). The increasing trend in
recirculating water in pulp mills to accomplish environmental demands is
aggravating these problems.
Therefore, the main objective of this work is to perform a thorough chemical
characterization of tagasaste trimming residues, with especial emphasis in the
chemical composition of lignin and lipophilic extractives. In this work, the
lignin composition of tagasaste was characterized in situ using analytical
pyrolysis coupled to gas chromatography/mass spectrometry (Py-GC/MS), a
powerful analytical tool for the rapid analysis of complex polymer mixtures
including lignocellulosic materials (Ralph and Hatfield 1991; Faix et al. 1990;
del Ro et al. 2001; del Ro et al. 2005) that can give information on the lignin
composition in terms of the H, G and S moieties. On the other hand, the
chemical characterization of the lipophilic extractives was performed by GC and
GC/MS by using high-temperature short- and medium-length capillary columns,
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5. Resultados y discusin
respectively. This method enables the elution and analysis of intact high
molecular weight lipids (Gutirrez et al. 1998). The knowledge of the chemical
composition of the main components of tagasaste trimming residues will be
useful for a better utilization of this agricultural waste.
2. Experimental
2.1. Samples
Tagasaste trimming residues were supplied by University of Huelva, Spain. The
dried samples were milled using a knife mill. For the isolation of lipids, the
milled samples were extracted with acetone in a Soxhlet apparatus for 8 h. The
acetone extracts were evaporated to dryness and resuspended in chloroform for
chromatographic analysis of the lipophilic fraction. Two replicates were used for
each sample, and all samples were subjected to GC and GC/MS analyses. For
Klason lignin content estimation, the samples extracted with acetone were
subsequently extracted with hot water (3 h at 100 C) to remove the water-
soluble material. Holocellulose was isolated from the pre-extracted fibers by
delignification for 4 hours using the acid chlorite method (Browning, 1967). The
D-cellulose content was determined by removing the hemicelluloses from the
holocellulose by alkali extraction (Browning, 1967). Klason lignin was
estimated as the residue after sulfuric acid hydrolysis of the pre-extracted
material according to Tappi rule T222 om-88 (Tappi, 1993). The acid-soluble
lignin was determined, after filtering off the insoluble lignin, by
spectrophotometric determination at 205 nm wavelength. Ash content was
estimated as the residue after 6 h at 575 C.
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5. Resultados y discusin
with a 1-min hold and then raised to a final temperature of 350 C at 15 C/min,
and held for 3 min. The injector and flame-ionization detector temperatures were
set at 300 and 350 C, respectively. Helium was used as the carrier gas at a rate
of 5 mL/min, and the injection was performed in splitless mode. Peaks were
quantified by area, and a mixture of standards (octadecane, palmitic acid,
sitosterol and cholesteryl oleate) was used to elaborate calibration curves.
The GC/MS analyses were performed with a Varian model Star 3400 GC
equipped with an ion trap detector (Varian Saturn 2000) using a medium-length
(12 m) capillary column of the same characteristics described above. The oven
was heated from 120 (1 min) to 380 C at 10 C/min and held for 5 min. The
transfer line was kept at 300 C. The injector was temperature programmed from
120 (0.1 min) to 380 C at a rate of 200 C/min and held until the end of the
analysis. Helium was used as the carrier gas at a rate of 2 mL/min.
Trimethylsilyldiazomethane methylation and BSTFA (bis(trimethylsilyl)-
trifluoroacetamide) silylation, in the presence of pyridine, were used to produce
the appropriate derivatives, when required. Compounds were identified by
comparing their mass spectra with mass spectra in Wiley and NIST libraries, by
mass fragmentography, and when possible, by comparison with authentic
standards.
2.4. Py-GC/MS.
The pyrolysis of tagasaste (1 mg) was performed in a micro-furnace pyrolyzer
(model 2020, Frontier Laboratories Ltd) directly connected to a GC/MS system
Agilent 6890 equipped with a fused silica capillary column HP 5MS
(30 m 0.25 mm 0.25 m I.D.). The detector consisted of an Agilent 5973
mass selective detector. The pyrolysis was performed at 500 C. The final
temperature was achieved at a rate of 20 C/min. The GC/MS conditions were as
follows: oven temperature was held at 50 C for 1 min and then increased up to
100 C at 30 C/min, from 100 to 300 C at 10 C/min and isothermal at 300 C
for 10 min using a heating rate of 20 C/min in the scan modus. The carrier gas
used was helium with a controlled flow of 1 ml/min. The compounds were
identified by comparing the mass spectra obtained with those of the Wiley and
NIST computer libraries and that reported in the literature (Faix et al. 1990;
Ralph and Hatfield 1991). Relative peak molar areas were calculated for
carbohydrate and lignin pyrolysis products. The summed molar areas of the
relevant peaks were normalized to 100%, and the data for two repetitive
pyrolysis experiments were averaged.
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5. Resultados y discusin
et al., 2004). This low lignin content is similar to that found in eucalypt wood
(Rencoret et al. 2007), a widely raw material for pulp and papermaking, and
together with the high holocellulose and D-cellulose contents makes tagasaste an
interesting raw material for pulp and paper production, as already advanced by
other authors (Daz et al. 2004; Lpez et al., 2004; Jimnez et al., 2006; Jimnez
et al. 2007; Garca et al., 2007). On the other hand, the acetone extractives
accounted for 1.4% but the chloroform-soluble lipids accounted for only 0.2%,
which is a value lower than that found in most lignocellulosic materials (Back
and Allen, 2000). However, while the content of the main organic fractions is an
important parameter in wood processing for pulp and papermaking, the chemical
composition of these organic fractions, in particular the lignin composition in
terms of the relative proportions of the S- and G-units, and the lipid
composition, in terms of the presence of saponifiable or unsaponifiable
components, strongly influences the pulping and bleaching behavior of a raw
material.
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5. Resultados y discusin
Table 1.- Identification and relative molar abundance of the compounds released by Py-
GC/MS of tagasaste.
No. Compound Origin Mass fragments MW %
1 acetic acid C 45/60 60 39.4
2 (3H) furan-3-one C 55/84 84 0.7
3 1,3-hydroxydihydro-2-(3H)-furanone C 58/102 102 3.1
4 (3H) furan-3-one C 55/84 84 1.0
5 2-furaldehyde C 67/95/96 96 3.8
6 2-(hydroxymethyl)-furan C 43/70/81/98 98 0.5
7 cyclopent-1-ene-3,4-dione C 54/68/96 96 0.6
8 (5H) furan-2-one C 55/84 84 2.9
9 2,3-dihydro-5-methylfuran-2-one C 55/69/98 98 3.1
10 5-methyl-2-furfuraldehyde C 53/109/110 110 0.4
11 4-hydroxy-5.6-dihydro-(2H)-pyran-2-one C 58/85/114 114 2.6
12 3-hydroxy-2-methyl-2-cyclopenten-1-one C 55/85/112 112 0.6
13 2-hydroxy-3-methyl-2-cyclopenten-1-one C 55/85/112 112 0.3
14 dihydroxypyran-1-one C 56/84/114 114 0.8
15 guaiacol LG 81/109/124 124 1.9
16 dimethyldihydropyranone C 55/70/83/111/126 126 0.3
17 4-methylguaiacol LG 95/123/138 138 1.2
18 catechol C 64/81/92/110 110 1.1
19 5-hydroxymethyl-2-tetrahydrofuraldehyde-3-one C 57/69/85/98/99 138 0.4
20 5-hydroxymethyl-2-furaldehyde C 69/97/109/126 126 0.3
21 methoxycatechol L 60/97/125/140 140 0.9
22 4-ethylguaiacol LG 122/137/152 152 0.4
23 4-vinylguaiacol LG 107/135/150 150 2.1
24 syringol LS 111/139/154 154 3.6
25 eugenol LG 131/149/164 164 0.4
26 4-propylguaiacol LG 122/136/166 166 0.1
27 vanillin LG 109/151/152 152 0.7
28 cis-isoeugenol LG 131/149/164 164 0.4
29 4-methylsyringol LS 125/153/168 168 1.8
30 trans-isoeugenol LG 131/149/164 164 1.2
31 homovanillin LG 122/137/166 166 0.2
32 4-propinylguaiacol LG 77/91/119/147/162 162 1.0
33 4-propinylguaiacol isomer LG 77/91/119/147/162 162 1.3
34 acetovanillone LG 123/151/166 166 0.2
35 4-ethylsyringol LS 167/182 182 1.5
36 guaiacylacetone LG 122/137/180 180 0.7
37 levoglucosane C 60/98 162 4.8
38 4-vinylsyringol LS 137/165/180 180 4.2
39 4-allylsyringol LS 167/179/194 194 0.7
40 cis-4propenylsyringol LS 167/179/194 194 0.5
41 syringaldehyde LS 167/181/182 182 1.2
192
5. Resultados y discusin
%G 38
%S 62
S/G 1.6
C: cellulose; LG: lignin guaiacyl; LS: lignin syringyl
38
24
5
44
3
23
11 15
9
29
8
30
2 13 4142 49
17 35
32 33
36 39 47
6 25 27 40 45 46
10 21
14 16 18 22 28 48
2 4 6 8 10 12 14 16 18 20 22
Retention time (min)
Figure 1. Py-GC/MS of tagasaste wood. The identities of the peaks are shown in Table 2.
193
5. Resultados y discusin
of the main lipid classes identified are summarized in Table 2. The structures of
the main lipophilic compounds identified in the tagasaste extracts are shown in
Figure 3. The main lipids identified in tagasaste were series of fatty acids,
including D-hydroxy acids, and steroid compounds, including steroid
hydrocarbons, steroid ketones, sterols, sterol esters and sterol glycosides. Other
compounds, such as series of alkanes and monoglycerides were also found in
minor amounts.
The series of free fatty acids (141.4 mg/kg) were present in the range from n-
tetradecanoic (C14) to n-hexacosanoic (C26) acids, with a strong even-over-odd
carbon atom predominance. Hexadecanoic (palmitic) acid (I, C16:0) and
octadecanoic (stearic) acid (C18:0) were the most abundant fatty acids followed
by n-tetracosanoic (C24) acid. Unsaturated fatty acids were also present, 9-
octadecenoic (oleic) acid (II, C18:1) being especially abundant. Fatty acids also
included a series of D-hydroxyfatty acids (51.8 mg/kg) that was present in the
range from 2-hydroxyoctadecanoic acid (C18) to 2-hydroxyhexacosanoic acid
(C26) with maximum at C24 (III), and the presence of exclusively the even
carbon atom number homologs.
34+5
steroid 2 steroid
hydrocarbons ketones
FA16
FA18:1
FA18
1
78
6
FA20 FA22 FA24
sterol esters
5 10 15 20
Retention time (min)
Figure 2. GC/MS of the lipophilic extracts from tagasaste wood. Key labels are: Fn: fatty
acids; 1: D-tocopherol; 2: campesterol; 3: stigmasterol; 4: sitosterol; 5: stigmastanol; 6: 7-
oxocampesterol; 7: 7-oxostigmasterol; 8: 7-oxositosterol.
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5. Resultados y discusin
Table 2.- Composition and abundance (mg/Kg) of lipophilic extractives in tagasaste trimming
residues.
compound mass fragments MW abundance
n-alkanes 8.8
n-eicosane 57/71/85/282 282 0.1
n-heneicosane 57/71/85/296 296 0.2
n-docosane 57/71/85/310 310 0.1
n-tricosane 57/71/85/324 324 0.3
n-tetracosane 57/71/85/338 338 0.2
n-pentacosane 57/71/85/352 352 0.5
n-hexacosane 57/71/85/366 366 0.3
n-heptacosane 57/71/85/380 380 0.5
n-octacosane 57/71/85/394 394 0.3
n-nonacosane 57/71/85/408 408 1.2
n-triacontane 57/71/85/422 422 0.2
n-hentriacontane 57/71/85/436 436 4.8
n-tritriacontane 57/71/85/464 464 0.1
fatty acids 141.4
n-tetradecanoic acid 60/73/228 228 1.1
n-pentadecanoic acid 60/73/242 242 1.0
9-hexadecenoic acid 55/69/236/254 254 2.2
n-hexadecanoic acid 60/73/129/256 256 55.4
n-heptadecanoic acid 60/73/129/270 270 2.9
9-octadecenoic acid 55/69/264 282 29.4
n-octadecanoic acid 60/73/129/284 284 10.7
n-nonadecanoic acid 60/73/129/298 298 1.4
n-eicosanoic acid 60/73/129/312 312 7.4
n-heneicosanoic acid 60/73/129/327 327 1.1
n-docosanoic acid 60/73/129/340 340 5.4
n-tricosanoic acid 60/73/129/354 354 6.0
n-tetracosanoic acid 60/73/129/368 368 10.3
n-pentacosanoic acid 60/73/129/382 382 2.6
n-hexacosanoic acid 60/73/129/396 396 4.5
-hydroxy fatty acids# 51.8
2-hydroxyoctadecanoic acid 73/327/371 386 0.6
2-hydroxyeicosanoic acid 73/355/399 414 0.9
2-hydroxydocosanoic acid 73/383/427 442 22.1
2-hydroxytetracosanoic acid 73/411/455 470 26.3
2-hydroxyhexacosanoic acid 73/439/483 498 1.9
steroid hydrocarbons 30.1
ergostatriene 135/143/380 380 5.6
ergostadiene 81/147/367/382 382 3.4
stigmasta-3,5,22-triene 135/143/394 394 11.7
195
5. Resultados y discusin
Steroid compounds were the second most important lipid class found among
the tagasaste extractives and included steroid hydrocarbons, steroid ketones and
sterols (in free and conjugated form). Sterols, in free and conjugated (esters and
glycosides) form were the most important steroids identified, free sterols being
present in important amounts (113.1 mg/kg). Stigmasterol (IV) was the most
abundant among the free sterols with the presence of important amounts of
campesterol (V) and sitosterol (VI) and minor amounts of stigmastanol (VII) as
well as the presence of the oxidized 7-ketocampesterol, 7-ketostigmasterol and
7-ketostigmastanol. Lower amounts of sterols could also be found in ester form
(28.3 mg/kg), with a predominance of sitosterol esters. Sterol glycosides, such
as campesteryl, stigmasteryl and sitosteryl 3E-D-glucopyranosides (VIII) were
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5. Resultados y discusin
also identified in lower amounts (13.2 mg/kg), the latter being the most
predominant. The identification of steryl glycosides was accomplished, after
BSTFA derivatization of the lipid extract, by comparison with the mass spectra
and relative retention times of authentic standards (Gutirez and del Ro, 2001).
It is important to point out the low content of free and conjugated (esters and
glycosides) sterols, in comparison to that found in eucalypt wood (Rencoret et
al., 2007), since these are the main compounds responsible for pitch deposition
during kraft cooking of hardwoods, such as eucalypt wood (del Ro et al. 1998,
2000; Silvestre et al. 1999; Gutirrez and del Ro 2001). On the other hand,
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5. Resultados y discusin
steroid ketones were also found in important amounts (30.5 mg/kg), being
mainly constituted by stigmasta-7,22-dien-3-one, stigmasta-3,5-dien-7-one (IX),
stigmast-4-en-3-one and stigmasta-3,6-dione. Different steroid hydrocarbons
(di- and triunsaturated) were also identified, although in low amounts (30.1
mg/kg), stigmasta-3,5,22-triene (X) being the most predominant.
Finally, a series of n-alkanes ranging from n-eicosane (C20) to n-tritriacontane
(C33) with n-hentriacontane (X, C31) being the most prominent, was found in low
amounts (8.8 mg/kg). And minor amounts of monoglycerides (8.1 mg/kg), such
as 1-monopalmitin (XI) and 1-monostearin, were also present among tagasaste
extractives.
4. Conclusions
Tagasaste is characterized by a high content of holocellulose and D-cellulose
and a relatively low lignin content. Moreover, the chemical composition of
tagasaste lignin indicates a predominance of S-lignin units over the G-lignin
ones (S/G molar ratio of 1.6) and the absence of H-lignin units. The total
acetone extractives accounted for 1.4% but the content on lipophilic compounds
is very low (only 0.2%). The main lipids identified in tagasaste were series of
fatty acids, including D-hydroxyfatty acids, and steroid compounds, including
steroid hydrocarbons, steroid ketones, sterols, sterol esters and sterol glycosides.
Acknowledgements
This study has been supported by the Spanish project AGL2005-01748. G.
Marques acknowledges a FPI Fellowship from the Spanish Ministry of
Education and Science. We thank Manuel J. Daz (University of Huelva, Spain)
for providing the tagasaste samples.
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A.T., 2005. Determining the influence of eucalypt lignin composition in paper
pulp yield using Py-GC/MS. J. Anal. Appl. Pyrolysis 74, 110-115.
Daz, M.J., Alfaro, A., Garca, M.M., Eugenio, F.M.E., Ariza, J., Lpez, F.,
2004. Ethanol pulping from tagasaste (Chamaecytisus proliferus L.F. ssp.
Palmensis). A new promising source for cellulose pulp. Ind. Eng. Chem. Res.
43, 1875-1881.
Faix, O., Meier, D., Fortmann, I., 1990. Thermal degradation products of wood.
A collection of electron of electron-impact (EI) mass spectra of monomeric
lignin derived products. Holz Roh- Werkst. 48, 351-354.
Garca, M.M., Lpez, F., Alfaro, A., Ariza, J., Tapias, R., 2007. The use of
tagasaste (Chamaecytisus proliferus) from different origins for biomass and
paper production. Biores. Technol. (in press).
Gonzlez-Vila, F.J., Almendros, G., del Ro, J.C., Martn, F., Gutirrez, A.,
Romero, J. 1999. Ease of delignification assessment of wood from different
Eucalyptus species by pyrolysis (TMAH)-GC/MS and CP/MAS 13C NMR
spectrometry. J. Anal. Appl. Pyrol. 49, 295-305.
Gutirrez, A., del Ro, J.C., Gonzlez-Vila, F.J., Martn, F., 1998. Analysis of
lipophilic extractives from Wood and pitch deposits by solid-phase extraction
and gas chromatography. J. Chromatogr. A 823, 449-455.
199
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Hillis, W.E., Sumimoto, M., 1989. Effect of extractives on pulping. In: Rowe,
J.W. (Ed.), Natural Products of Woody Plants II; Springer-Verlag, Berlin. pp.
880-920.
Jimnez, L., Prez, A., Rodrguez, A., de La Torre, M.J., 2006. New raw
materials and pulping processes for production of pulp and paper. Afinidad 63
(525), 362-369.
Jimnez, L., Prez, A., de la Torre, M.J., Moral A., Serrano, L., 2007.
Characterization of vine shoots, cotton stalks, Leucaena leucocephala and
Chamaecytisus proliferus, and of their ethyleneglycol pulps. Biores. Technol.
98, 3487-3490.
Kindu, M., Glatzel, G., Tadesse, Y., Yosef, A., 2006. Tree species screened on
nitosols of central Ethiopia: Biomass production, nutrient contents and effect
on soil nitrogen. J. Trop. Forest Sci. 18, 173-180.
Lpez, F., Alfaro, A., Garca, M.M., Diaz, M.J., Calero, A.M., Ariza, J., 2004.
Pulp and paper from tagasaste (Chamaecytisus proliferus LF ssp palmensis).
Chem. Eng. Res. & Des. 82, 1029-1036.
Rencoret, J., Gutirrez, A., del Ro, J.C., 2007. Lipid and lignin composition of
woods from different eucalypt species. Holzforschung 61, 165-174.
Silvestre, A.J.D., Pereira, C.C.L., Pascoal Neto, C., Evtuguin, D.V., Duarte,
A.C., Cavaleiro, J.A.S., Furtado, F.P., 1999. Chemical composition of pitch
deposits from an ECF Eucalyptus globulus bleached kraft pulp mill: its
relationship with wood extractives and additives in process streams. Appita J.
52, 375-382.
Technical Association of the Pulp and Paper Industry, 1993. Test methods,
1992-1993. TAPPI, Atlanta, GA..
Tsutsumi, Y., Kondo, R., Sakai, K., Imamura, H., 1995. The difference of
reactivity between syringyl lignin and guaiacyl lignin in alkaline systems.
Holzforschung 49, 423-428.
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Ventura, M.R., Castanon, J.I.R., Rey, L., Flores, M.P., 2002. Chemical
composition and digestibility of tagasaste (Chamaecytisus proliferus)
subspecies for goats. Small Ruminant Res. 46, 207-210.
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Publicacin VII:
Marques G., del Ro J.C. and Gutirrez A. (2010) Lipophilic extractives from
several nonwoody lignocellulosic crops (flax, hemp, sisal, abaca) and their fate
during alkaline pulping and TCF/ECF bleaching. Bioresource Technology, 101,
260-267.
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5. Resultados y discusin
Instituto de Recursos Naturales y Agrobiologa, CSIC, PO Box 1052, E-41080, Seville, Spain
Abstract
The fate of lipophilic extractives from several nonwoody species (flax, hemp,
sisal and abaca) used for the manufacturing of cellulose pulps, was studied
during soda/anthraquinone (AQ) pulping and totally chorine free (TCF) and
elemental chlorine free (ECF) bleaching. With this purpose, the lipophilic
extracts from the raw materials and their unbleached and bleached industrial
pulps, were analyzed by gas chromatography-mass spectrometry. Aldehydes,
hydroxyfatty acids and esterified compounds such as ester waxes, sterol esters
and alkylferulates strongly decreased after soda/AQ pulping while alkanes,
alcohols, free sterols and sterol glycosides survived the cooking process. Among
the lipophilic extractives that remained in the unbleached pulps, some amounts
of free sterols were still present in the TCF pulps whereas they were practically
absent in the ECF pulps. Sterol glycosides were also removed after both TCF
and ECF bleaching. By contrast, saturated fatty acids, fatty alcohols and alkanes
were still present in both bleached pulps.
Keywords: pitch; lipophilic extractives; paper pulp; nonwoody fibers
1. Introduction
Lipophilic extractives, i.e. the non-polar extractable fraction from wood and
other lignocellulosic crops, include different classes of compounds, such as
alkanes, fatty alcohols, fatty acids, free and conjugated sterols, terpenoids,
triglycerides and waxes. These lipophilic compounds, even when present in low
amounts in the raw material, may play an important role in industrial wood
processing for bleached pulp and paper production since they are at the origin of
the so-called pitch deposits along the pulp and paper manufacturing processes.
Pitch deposition is a serious problem in the pulp and paper industry since it is
responsible for reduced production levels, higher equipment maintenance costs,
higher operating costs, and an increased incidence of defects in the finished
products, which reduces quality and benefits (Back and Allen, 2000).
The nature and severity of pitch deposition depend not only on the raw
materials used (and hence on the nature of the lipophilic compounds), but also
on the industrial processes of pulping and bleaching applied at the mill. In the
manufacture of alkaline pulps, a large part of the lipids originally present in the
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5. Resultados y discusin
raw material is removed during the cooking and bleaching stages. However,
some chemical species survive these processes and are found as pulp extractives
(Bergelin and Holmbom, 2003; Freire et al., 2005; 2006; Gutirrez et al.,
2001a), suspended in process waters (Gutirrez et al., 2001b) or forming the so-
called pitch deposits in circuits, equipments and final product (Bergelin et al.,
2005; del Ro et al., 1998; 2000; Gutirrez and del Ro, 2005; Silvestre et al.,
1999). Growing pressure for closing water loops in pulp and paper mills leads to
increasing build up of lipophilic compounds in the processes and therefore, to an
increasing number of severe and costly pitch related problems.
The different classes of lipids have different behavior during pulping and
bleaching. Several studies have provided information on the behavior of wood
lipids, such as fatty and resin acids, triglycerides and sterols and triterpenols
during pulping and bleaching (Back and Allen, 2000; Bergelin and Holmbom,
2003; 2008; Freire et al., 2005; 2006; Gutirrez et al., 2001a; Shin and Kim,
2006). However, the chemistry of lipids from nonwoody pulps in pulping and
bleaching has not been examined much so far (Gutirrez et al., 2004; Gutirrez
and del Ro, 2003a; 2003b). Likewise, to the best of our knowledge, there is
very little information available dealing with pitch problems on nonwoody pulps
(Gutirrez and del Ro, 2005). In this context, the aim of this work was to
identify the specific lipophilic constituents of different nonwoody fibers, which
are used for the manufacturing of cellulose pulps for specialty papers, and to
study their behavior during soda/anthraquinone (AQ) pulping and totally chorine
free (TCF) and elemental chlorine free (ECF) bleaching. For this, a series of
pulps (crude pulps taken after soda/AQ pulping and bleached pulps taken after
TCF and ECF bleaching) from different nonwoody raw materials (flax, hemp,
sisal, abaca) were selected for this study. The composition of the lipophilic
compounds in the fibers and their respective pulps was analyzed by gas
chromatography (GC) and gas chromatography-mass spectrometry (GC-MS)
using short- and medium-length high temperature capillary columns,
respectively, with thin films, which enables the elution and analysis of intact
high molecular weight lipids such as waxes or sterol glycosides (Gutirrez et al.,
1998; Gutirrez and del Ro, 2001). The knowledge of the behavior of lipophilic
extractives during pulping and bleaching is an important step towards
understanding and predicting the pitch problems and designing effective
solutions for its control.
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5. Resultados y discusin
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5. Resultados y discusin
Fatty alcohols, fatty acids and sterol glycosides were determined as silylated
derivatives in the derivatized samples. Hydroxyfatty acids were determined in
the samples after methylation followed by silylation. The rest of compounds
were determined in the underivatized samples. Peaks were quantified by area
and a mixture of standards (octadecane, palmitic acid, sitosterol, cholesteryl
oleate, and sitosteryl 3-D-glucopyranoside) with a concentration range between
0.1 and 1mg/ml was used to elaborate calibration curves. The data from the two
replicates were averaged.
Table 1. Lipophilic extractives content (%) in the selected nonwoody raw materials and
pulps
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5. Resultados y discusin
OH
I II
O O
H OH
III IV
HO O
V VI
CH2OH
O
OH O HO
HO
VII VIII
OH
O
IX
The main lipid classes present in the nonwoody fibers selected for this study
were series of alkanes (I), fatty alcohols (II), aldehydes (III), fatty acids (IV),
steroids, including free (V) and conjugated sterols (VI-VII), triterpenoids (VIII)
and ester waxes (IX). The detailed composition of the lipophilic compounds
present in these fibers have been previously addressed (del Ro et al., 2004; del
Ro and Gutirrez, 2006; Gutirrez et al., 2006; 2008; Gutirrez and del Ro,
2003a; 2003b).
In the case of flax bast fibers the predominant lipophilic compounds present
were series of fatty acids and aldehydes, accounting for 34% and 23% of total
extract, respectively, followed by ester waxes (18%), and fatty alcohols (13%).
Fatty acids were also the predominant compounds (27% of total extracts) in
hemp bast fibers, followed by alkanes (15%), free sterols (12%) and steroid
hydrocarbons (12%). Among the selected leaf fibers, free sterols predominated
in both sisal (20%) and abaca (45%), followed by alkanes (14%) and fatty acids
(10%) in sisal, and by fatty acids (16%) in abaca fibers.
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5. Resultados y discusin
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5. Resultados y discusin
Ak 29 5+6+7
+
Al26 11
Hemp bast fibers
2
1 9
Fa18:1 +
+ 10
Fa16 Fa18:2 Ak 27 8
+ 34 W44
Fa18 Al24 12 W42 W46
SE W48 W50
5+6+7
Ak 31
+
Al28 Crude pulp
Ak 29
+
Al26
Fa18:1 4 Al30
+ Ak 27 3
Fa18:2
AQ + 11
Al24
Fa16 Fa18 1
5+6+7
Ak 29 Ak 31
+ +
Al26 Al28 TCF pulp
Def oamer
Fa16 Al30
Fa18
Ak 27 11
AQ
+ 1 34
Al24
Ak 31
+
Al28
Ak 29
+
Al26
ECF pulp
Al30
11
7
Ak 27
Al22 +
AQ Al24
Fa16 Fa18 1
5 10 15 20 25
Retention time (minutes)
Fig. 2. GC-MS chromatograms of underivatized lipophilic extractives from hemp fibers (raw
material), and their crude, TCF and ECF pulps. Peak identification, 1: stigmasta-3,5,22-triene;
2: stigmasta-3,5-diene; 3: campesterol; 4: stigmasterol; 5: sitosterol; 6: stigmastanol; 7: -
amyrin; 8: stigmasta-3,5-dien-7-one; 9: -amyrin acetate; 10: stigmast-4-en-3-one; 11:
friedelan-3-one; 12: stigmastane-3,6-dione; AQ: anthraquinone; Fa(n): fatty acids; Ak(n):
alkanes; Al(n): fatty alcohols; SE: sterol esters; W(n): ester waxes; n denotes the total carbon
atom number.
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5. Resultados y discusin
5+6+7
Al28
1 4
Ak 29
+ 2 8
Al26
Fa16 3
Ak 25 F24 9
Ak 21 Ak 23 Ak 27
Fe26 Fe28
Fe24
5+6+7
Al28
Crude pulp
Ak 29
+
Al26 4
AQ Ak 25 Ak 27
Al30
Fa16 + + 3 9
Al22 Al24 1 8
Fa18
Al28
5+6
Ak 29
+ TCF pulp
Al26
Def oamer Ak 25 4
+ Ak 27
Al22 + Al30
Al24 9
AQ Fa16 Fa18 1 3
Al28
Ak 29
+
Al26
5+6 ECF pulp
Ak 25 Ak 27 Al30
Fa16 + +
Al24 4
AQ Al22
1 9
Fa18
5 10 15 20 25
Retention time (minutes)
Fig. 3. GC-MS chromatograms of underivatized lipophilic extractives from sisal fibers (raw
material), and their crude pulp, TCF pulp and ECF pulps. Peak identification, 1: stigmasta-
3,5,22-triene; 2: stigmasta-3,5-diene; 3: campesterol; 4: stigmasterol; 5: sitosterol; 6:
stigmastanol; 7: -amyrin; 8: stigmasta-3,5-dien-7-one; 9: hecogenin; AQ: anthraquinone;
Fa(n): fatty acids; Ak(n): alkanes; Al(n): fatty alcohols; Fe(n): n-alkylferulates; n denotes the
total carbon atom number.
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5. Resultados y discusin
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5. Resultados y discusin
* Abreviations: Glcp (glucopyranoside); C(n), n denotes the total carbon atom number; tr: traces
212
5. Resultados y discusin
Finally, alkanes, fatty alcohols and free sterols and triterpenols survived the
alkaline conditions and therefore were the main lipophilic constituents in crude
pulps.
The behavior of the fatty acids in an aqueous environment is quite different
from that of the neutrals. At alkaline pH, the acids dissociate and can dissolve in
water to quite a high extent forming fatty acid soaps that can form micelles.
Fatty acid soaps are effective solubilizing agents facilitating the removal from
pulp of sparingly soluble neutral substances (Back, 2000). The ratio of
saponifiables-to-unsaponifiables has been suggested to be a better index for
predicting pitch problems than the total amount of lipids (Back and Allen,
2000). In fact, the higher abundances of unsaponifiable compounds with respect
to the saponifiable ones is the main cause for pitch problems during pulping of
some woods, such as aspen or eucalypt (Chen et al., 1995; del Ro et al., 1998;
2000). This fact can explain why the lipid content of crude flax pulps is similar
or even lower than the other pulps regardless flax fibers had the highest
extractive content. As mentioned above, fatty acids are the predominat lipids in
flax fibers and can help to solubilize other water-insoluble components such as
fatty alcohols and sterols. In contrast, the lipid content of crude sisal pulp is
higher than the other three pulps regardless the relatively low content of
lipophilic extractives in the raw material. Sisal fibers have low fatty acid content
whereas the content in neutrals such as alkanes, fatty alcohols and steroids is
relatively high. Particularly, the high abundances of free sterols, which have a
high propensity to form pitch deposits (del Ro et al., 1998; 2000), in the crude
pulps from hemp and sisal, would point to a high pitch deposition tendency of
the lipophilics from these fibers.
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5. Resultados y discusin
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5. Resultados y discusin
compared to ECF bleaching using chlorine dioxide (Back and Allen, 2000;
Gutirrez et al., 2009) may cause pitch problems to be, in principle, more severe
in the former bleaching sequences. This is specially evident in the case of
unsaturated steroids and triterpenoids as well as unsaturated fatty acids, which
are strongly modified by chlorine dioxide but remain practically unaltered by
oxygen and hydrogen peroxide (Holmbom, 2000; Bergelin and Holmbom, 2003;
Freire et al., 2005; 2006; Gutirrez et al., 2001a). However, in the nonwoody
pulps studied here, the major lipophilic compounds present are saturated fatty
acids, fatty alcohols, alkanes, which do not show reactivity towards chlorine
dioxide, and therefore there are not great differences in the composition of the
lipophilic extractives between ECF and TCF bleached pulps, with the exception
of abaca pulp which lacks fatty alcohols and alkanes and where unsaturated
sterols are the predominant lipophilic compounds. Therefore, both ECF and TCF
bleached pulps will undergo similar pitch problems. Fatty acids, fatty alcohols
and alkanes have been reported to be the compounds responsible for pitch
deposits formed during pulping of nonwoody plants (Gutirrez and del Ro,
2005).
4. Conclusions
A thorough chemical characterization of the lipophilic extractives from different
nonwoody fibers (flax, hemp, sisal and abaca) at different stages of pulp
production (soda/AQ pulping and TCF/ECF bleaching) has been carried out.
This study provides useful information into the extent of their removal along the
cooking and bleaching processes. The soda/AQ pulping stage led to the removal
of aldehydes, hydroxyfatty acids and the complete hydrolysis of esterified
compounds such as ester waxes, sterol esters and alkyl ferulates. Among the
bleaching processes, ECF bleaching showed high reactivity towards unsaturated
sterols and both ECF and TCF bleaching were very effective in removing sterol
glycosides from nonwoody pulps. In contrast, saturated fatty acids, fatty
alcohols and alkanes, which are the main lipophilic compounds in most of the
studied fibers, survived pulping and bleaching conditions and were the
predominating compounds in both TCF and ECF bleached pulps.
Acknowledgements
This study has been supported by the Spanish projects BIO2007-28719-E and
AGL2008-00709 and the EU project BIORENEW (NMP2-CT-2006-026456).
We thank CELESA pulp mill (Tortosa, Spain) for providing the samples. G.M.
thanks the Spanish MEC for a FPI fellowship.
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samples in kraft pulp production. Pap. Puu-Pap. Tim. 87, 399-403.
Chen, T., Wang, Z., Zhou, Y., Breuil, C., Aschim, O.K., Yee, E., Nadeau, L.,
1995. Using solid-phase extraction to assess why aspen causes more pitch
problems than softwoods in kraft pulping. Tappi J. 78, 143-149.
del Ro, J.C., Gutirrez, A., 2006. Chemical composition of abaca (Musa
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Food Chem. 54, 4600-4610.
del Ro, J.C., Gutirrez, A., Gonzlez-Vila, F.J., Martn, F., Romero, J., 1998.
Characterization of organic deposits produced in the kraft pulping of
Eucalyptus globulus wood. J. Chromatogr. A 823, 457-465.
del Ro, J.C., Rodrguez, I.M., Gutirrez, A., 2004. Identification of intact long-
chain p-hydroxycinnamate esters in leaf fibers of abaca (Musa textilis) using
gas chromatography/mass spectrometry. Rapid Commun. Mass Sp. 18, 2691-
2696.
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del Ro, J.C., Romero, J., Gutirrez, A., 2000. Analysis of pitch deposits
produced in kraft pulp mills using a totally chlorine free bleaching sequence.
J. Chromatogr. A 874, 235-245.
Freire, C.S.R., Silvestre, A.J.D., Neto, C.P., Evtuguin, D.V., 2006. Effect of
oxygen, ozone and hydrogen peroxide bleaching stages on the contents and
composition of extractives of Eucalyptus globulus kraft pulps. Bioresource
Technol. 97, 420-428.
Gutirrez, A., del Ro, J.C., 2003a. Lipids from flax fibers and their fate in
alkaline pulping. J. Agr. Food Chem. 51, 4965-4971.
Gutirrez, A., del Ro, J.C., 2003b. Lipids from flax fibers and their fate in
alkaline pulping (Vol 51, pg 4965, 2003). J. Agr. Food Chem. 51, 6911-6914.
Gutirrez, A., del Ro, J.C., 2005. Chemical characterization of pitch deposits
produced in the manufacturing of high-quality paper pulps from hemp fibers.
Bioresource Technol. 96, 1445-1450.
Gutirrez, A., del Ro, J.C., Gonzlez-Vila, F.J., Martn, F., 1998. Analysis of
lipophilic extractives from wood and pitch deposits by solid-phase extraction
and gas chromatography. J. Chromatogr. A 823, 449-455.
Gutirrez, A., del Ro, J.C., Ibarra, D., Rencoret, J., Romero, J., Speranza, M.,
Camarero, S., Martnez, M.J., Martnez, A.T., 2006. Enzymatic removal of
free and conjugated sterols forming pitch deposits in environmentally sound
bleaching of eucalypt paper pulp. Environ. Sci. Technol. 40, 3416-3422.
Gutirrez, A., del Ro, J.C., Martnez, A.T., 2009. Microbial and enzymatic
control of pitch in the pulp and paper industry. Appl. Microbiol. Biot. 82,
1005-1018.
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5. Resultados y discusin
Gutirrez, A., Rodrguez, I.M., del Ro, J.C., 2004. Chemical characterization of
lignin and lipid fractions in kenaf bast fibers used for manufacturing high-
quality papers. J. Agr. Food Chem. 52, 4764-4773.
Gutirrez, A., Rodrguez, I.M., del Ro, J.C., 2006. Chemical characterization of
lignin and lipid fractions in industrial hemp bast fibers used for manufacturing
high-quality paper pulps. J. Agr. Food Chem. 54, 2138-2144.
Gutirrez, A., Rodrguez, I.M., del Ro, J.C., 2008. Chemical composition of
lipophilic extractives from sisal (Agave sisalana) fibers. Ind. Crop. Prod. 28,
81-87.
Gutirrez, A., Romero, J., del Ro, J.C., 2001a. Lipophilic extractives from
Eucalyptus globulus pulp during kraft cooking followed by TCF and ECF
bleaching. Holzforschung 55, 260-264.
Gutirrez, A., Romero, J., del Ro, J.C., 2001b. Lipophilic extractives in process
waters during manufacturing of totally chlorine free kraft pulp from eucalypt
wood. Chemosphere 44, 1237-1242.
Hillis, W.E., Sumimoto, M., 1989. Effect of extractives on pulping, in: Rowe, J.
W. (ed.), Natural products of woody plants. II. Springer-Verlag, Berlin, pp.
880-920.
Holmbom, B., 2000. Resin reactions and deresination in bleaching, in: Back, E.
L., Allen, L. H. (Eds.), Pitch control, wood resin and deresination. Tappi
Press, Atlanta, pp. 231-244.
Shin, S.J., Kim, C.H., 2006. Residual extractives in unbleached aspen and pine
kraft pulps and their fate on oxygen delignification. Nord. Pulp Pap. Res. J.
21, 260-263.
Silvestre, A.J.D., Pereira, C.C.L., Neto, C.P., Evtuguin, D.V., Duarte, A.C.,
Cavaleiro, J.A.S., Furtado, F.P., 1999. Chemical composition of pitch
deposits from ECF Eucalyptus globulus bleached kraft pulp mill: Its
relationship with wood extractives and additives in process streams. Appita J.
52, 375-382.
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Publicacin VIII:
Marques G., Gutirrez A., del Ro J.C. and Evtuguin D.V. (2010) Acetylated
heteroxylan from Agave sisalana and its behavior in alkaline pulping and
TCF/ECF bleaching. Carbohydrate Polymers, 81, 517-523.
220
5. Resultados y discusin
Abstract
The heteroxylan from sisal (Agave sisalana), an O-acetyl-(4-O-
methylglucurono)xylan with a molecular weight (Mw) of 18 kDa, was isolated
by extraction of peracetic holocellulose with Me2SO and thoroughly
characterized by wet chemistry, and NMR spectroscopy. The heteroxylan
backbone is composed of (14)-linked -D-xylopyranosyl units (Xylp)
partially branched with terminal (12)-linked 4-O-methyl--D-glucuronosyl
(MeGlcpA, 9%mol.) and a small proportion of -D-glucuronosyl (GlcpA,
<1%mol.) residues. Roughly 61%mol. of Xylp residues are acetylated (DS
=0.70). During soda/AQ pulping of sisal fibers, MeGlcpA and GlcpA are mostly
removed or converted to 4-deoxy--L-threo-hex-4-enopyranosyluronic acid
(HexA), although about 15% of the initially present MeGlcpA was maintained
intact upon cooking. The major part of acetyl groups (95%) was hydrolyzed
during pulping. It was proposed that during bleaching, a low molecular weight
xylan fraction associated to residual lignin was removed from pulp and small
proportion of MeGlcpA was additionally converted to HexA. The profiles of
uronosyl residues in xylans from TCF and ECF bleached sisal pulps were rather
different.
Keywords: Agave sisalana; soda pulping; bleaching; black liquor; xylan;
structural analysis; NMR spectroscopy
1. Introduction
Sisal fibers are hard fibrous material isolated from the leaves of the sisal plant
(Agave sisalana), a monocotyledonous plant from the Agavaceae family (Li et
al. 2000; Gutirrez et al. 2008). Originally from Central America and Mexico,
sisal plant is widely cultivated in South America (e.g. Brazil), Australia and
Africa (e.g. Kenya) (Gutirrez et al, 2008; Mwaikambo, 2006). The sisal fibers
find out numerous applications in the manufacture of ropes for boats, goods for
the agricultural industry and for the reinforcement of polymeric matrices (Li et
al., 2000; Gutirrez et al., 2008; Mwaikambo, 2006; Megiatto et al., 2007). Sisal
cellulosic pulp possesses such characteristics as high tear resistance, alpha
cellulose content, porosity, bulk, moister absorbency and high folding
endurance, that offer unique opportunities for the papermaking (Gutirrez et al.,
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5. Resultados y discusin
2008; Mwaikambo, 2006; Megiatto et al., 2007; Hurter, 2001; Idrraga et al.,
2001). Easily bleachable sisal chemical pulp is industrially produced by soda
pulping in the presence of athroquinone (AQ) as catalyst.
The basic knowledge of the chemical composition of sisal fibers, as well as
the behavior of its components during pulping and bleaching, is essential for the
better understanding and improving the pulping and bleaching operations and
for the assessment of pulp and paper properties. Previous papers have reported
the composition of the lipophilic compounds (Gutirrez et al., 2008) and the
structure of the lignin (del Ro et al., 2007, 2008) of sisal fibers, but only limited
work has been performed on the structural characterization of the carbohydrate
fraction of this fiber (Das Gupta & Mukherjee, 1967; Stewart et al., 1997;
Megiatto et al., 2007). In the present work, we report the structural
characterization of a heteroxylan in sisal fibers, as well as in their soda/AQ
pulps (unbleached and TCF/ECF bleached). The study of hemicelluloses is of
fundamental and practical interest, since their partial degradation and dissolution
during pulping is responsible for significant consumption of pulping chemicals,
the decrease of pulp yield and the papermaking properties of bleached pulps
(Evtuguin et al., 2003; Pinto et al., 2005; Lisboa et al., 2004).
Hemicelluloses provide a supporting function to the cell wall being bounded
to cellulose fibrils. Hemicelluloses are mainly branched polymers of low
molecular weight (DP 80-200) and are composed by diverse sugar residues
(D-xylose, L-arabinose, D-glucose, D-galactose, D-mannose, D-glucuronic acid,
4-O-methyl-D-glucuronic acid, D-galacturonic acid, and to a lesser extent, L-
rhamnose, L-fucose, and various O-methylated neutral sugars) (Shimizu, 1991;
Sun et al., 2000; Ebringerov et al., 2005). In particular, glucuronoxylan (GX) is
the major hemicellulose in such important industrial crops produced by agro-
industry as kenaf, bamboo, flax, sisal and jute and is structurally similar to
hardwood xylans (Rowell et al., 1998; Gorshkova et al., 1996; Neto et al., 1996;
Vignon & Gey, 1997; Stewart et al., 1997). Among the above mentioned plants,
GX of sisal is one of the less investigated. Previous structural/compositional
studies of GX from sisal were carried out mainly with alkali-extracted GX
(Stewart et al., 1997; Das Gupta & Mukherjee, 1967). Hence, some essential
structural information, such as substitution patterns with acetyl groups, was not
assessed. It was suggested, however, that sisal GX is an O-acetyl-4-O-
methylglucuronoxylan with moderate molecular weight, around 15-20 kDa
(Stewart et al., 1997; Das Gupta & Mukherjee, 1967). According to data from
methylation analysis of sisal xylan, its backbone is constituted by E-(1-4)-linked
D-xylopyranose residues, carrying a low degree of substitution (8-10 % mol.)
with terminal 4-O-methyl-D-glucopyranosyluronic acid residue linked through
the O-2 position (Das Gupta & Mukherjee, 1967).
In this report, the heteroxylan from sisal fibers was isolated by extraction of
peracetic holocellulose with dimethyl sulfoxide and thoroughly characterized by
wet chemistry and NMR spectroscopy. This isolation procedure allowed to
222
5. Resultados y discusin
obtain a xylan sample with intact O-acetyl moieties. Simultaneously, the fate of
this heteroxylan structure during soda/anthraquinone pulping and TCF and ECF
bleaching was also studied.
2. Experimental
2.1. Samples
Sisal (Agave sisalana) leaf fibers from Africa (Kenya), their soda/AQ chemical
pulps (unbleached and ECF/TCF bleached) and cooking black liquor were
supplied by CELESA pulp mill (Tortosa, Spain). The TCF bleaching sequence
E(O)P-EP included two hydrogen peroxide stages at 90C, the first under
oxygen pressure (E(O)P stage) and the second without oxygen (EP stage). The
ECF bleaching sequence D-EP included a chlorine dioxide (D) at 60C and a
hydrogen peroxide (EP) stages at 90C.
The samples were air-dried, milled using a knife mill (Janke and Kunkel,
Analysenmhle), and extracted with acetone in a Soxhlet apparatus for 8 h. For
estimation of the Klason lignin content, the acetone extracted samples were
subsequently extracted with hot water (3 h at 100 C). Klason lignin was
estimated as the residue after 72% sulfuric acid hydrolysis of the pre-extracted
material according to Tappi standard procedures (Tappi Test Methods, 1993).
Ash content was estimated as the residue after 6 h calcination at 575 C.
223
5. Resultados y discusin
224
5. Resultados y discusin
peaks were identified by comparing their mass spectra with mass spectra in
Wiley and NIST libraries and that reported in the literature (Sundberg et al.,
1996; Bertaud et al., 2002; Bleton et al., 1996).
225
5. Resultados y discusin
Table 1. Chemical composition of sisal fibers, unbleached soda pulp and ECF and TCF
bleached pulps (% w/w).
Component Sisal fibers Unbleached pulp TCF pulp ECF pulp
Ash 1.0 1.0 0.4 0.4
Extractives (acetone) 0.8 0.3 0.1 0.1
Extractives (water) 2.3 0.7 0.6 0.4
Klason lignin 5.9 0.7 - -
Holocellulose 70.0 95.0 - -
Cellulose* 54.5 - - -
Neutral sugars
Rha 0.7 0.7 tr tr
Ara 1.3 tr tr tr
Xyl 20.0 19.0 19.4 20.6
Man 0.8 - - -
Gal 1.0 tr tr tr
Fuc <0.5 - - -
Glc 75.7 80.4 80.6 79.4
* Krschner-Hoffer method of determination; tr: traces;
226
5. Resultados y discusin
Table 2. Neutral monosaccharide composition (% w/w) of xylans isolated from sisal fibers,
pulps and black liquor.
Rha Ara Xyl Man Fuc Gal Glc
Sisal fibers 0.9 0.7 93.7 tr - 1.2 3.5
Unbleached pulp tr tr 99.5 - - tr 0.5
TCF bleached pulp tr tr 99.0 - - tr 1.0
ECF bleached pulp tr tr 98.7 - - tr 1.3
Black liquor precipitated polysaccharides
(BLPS) 0.7 3.6 10.9 2.6 1.1 27.1 54.0
tr: traces
227
5. Resultados y discusin
Xyl
SISAL FIBERS
GalA
Gal
4-O-MeGLCA
GlcA
Xyl
Ara
Xyl
GalA
GalA
Ara
IS Dimers
GalA
Ara
Rha
Glc
Gal
Glc
Gal
Xyl
8 10 12 14 16 18 20 22 24 26
228
5. Resultados y discusin
1200
960
Response
720
14 kDa (D)
240 (B)
10 kDa
18 kDa (A)
0
Figure 2. The GPC elution curves of xylans isolated from sisal fibers and their pulps. (A)
sisal fibers, (B) unbleached pulp, (C) TCF bleached pulp, (D) ECF bleached pulp.
229
5. Resultados y discusin
X5 60
MeGlcA2
MeGlcA3 X3Ac2 X5 X2Ac3
70
X2Ac2
X3Ac3MeGlcA
X4Ac3MeGlcA X3 X2
X3Ac3 X4 80
X4Ac2
X2Ac3MeGlcA
MeGlcA4 90
X1Ac2
MeGlcA1
X1Ac3 100
X1Ac3MeGlcA
X1
230
5. Resultados y discusin
spectra (Figure 6). The presence of HexA, formed under alkaline pulping
conditions via E-elimination of methoxyl group, was confirmed applying the
total correlation spectroscopy (TOCSY), according to previously published
proton chemical shifts (Teleman et al. 1995). The HexA residues may be easily
detected in the anomeric region of 1H NMR spectra, which showed the
appearance of new signals at 5.36 and at 5.82 ppm that were assigned to H-1 and
H-4 in corresponding structures (Figure 6). The HexA content in sisal soda pulp
III
I
IV V IV
II
H1/H4 H3/H5
H1/H2 H1/H4
H3/H2 3.5
H1/H2
H1/H3 H3/H4
4.0
H3/H1 4.5
H3/H1 H1
H3/H1
H3 H1/H3 5.0
H1 H3
H3
5.5
5.5 5.4 5.3 5.2 5.1 5.0 4.9 4.8 ppm
III- 4)[3-O-Ac]--D-Xylp-(1
Figure 4. Anomeric region of the TOCSY spectrum (D2O, 60 C) of heteroxylan from sisal
fibers
231
5. Resultados y discusin
was 60.6 mmol/kg of pulp as determined after pulp treatment under acidic
conditions followed by detection of furoic acid derivatives by UV-spectroscopy
at 245 nm (Vuorinen et al. 1999).
The balance of uronic moieties in the initial xylan and in the xylan remaining
in the pulp was estimated based on the ratio of anomeric protons in uronic
groups at 5.27 ppm and in internal xylopyranose units at 4.47 ppm (pulp xylan)
or at 4.40-4.65 ppm (fiber xylan). This analysis indicated a removal of about
30% of all uronic units (MeGlcpA and HexA) from xylan during pulping.
The polysaccharides dissolved in the black liquor (BLPS) during pulping
were isolated according to a previously published procedure (Engstrm et al,
1995) and chemically characterized (Table 2). The aim of this study was to
compare the structure of the xylan remaining in the pulp with that dissolved in
the pulping liquor. Surprisingly, the heteroxylan was a minor polysaccharide
dissolved in the liquor and its purification by fractional precipitation failed. At
the same time, the analysis of neutral sugars of BLPS revealed glucans as the
major precipitated polysaccharides (glucose represents around 54 % of BLPS
weight) followed by galactans. The preliminary study on BLPS using multiple
bonds 1H-1H correlation NMR spectroscopy (TOCSY) gave additional insights
H2O CH3-CO-
*
H1/H2
Xyl-2Ac H1
H1
H3 Xyl-3Ac
Xyl-3Ac Xyl
H1 H3 Xyl-
MeGlcA 3Ac-2GlcA
H3
Xyl-2,3Ac
5.5 5.4 5.3 5.2 5.1 5.0 4.9 4.8 4.7 4.6 4.5 4.4
ppm
Figure 5. 1H NMR spectrum (D2O, 30 C) of heteroxylans from sisal fibers (top image) and
the expanded region of anomeric protons (bottom image). The designations for the structural
fragments are the same as in Table 5. * solvent impurities.
232
5. Resultados y discusin
H1 Xyl
H1
MeGlcA
H1
HexA
H4 HexA
Figure 6. 1H NMR spectra (D2O, 30 C ) of heteroxylans isolated from sisal unbleached pulp
and TCF and ECF bleached pulps.
into the type of glucans dissolved during pulping from sisal fibers (Figure 7).
These were suggested to be mixture of E-glucans, in particular, E-(13)-
glucans with a low degree of ramification at C6 (callose type), by comparison of
the proton-proton correlations with previously published data on proton
resonances in E-(13)-glucan (Torosantucci et al. 2005). However, a more
detailed study is required to elucidate the exact structure of the E-glucans in sisal
fibers.
233
5. Resultados y discusin
OH
OH H
H
H O
H O
HO O
HO O
O OH
OH H
H
H H
H6ax H5
H1 H4
H3 H2
3.50
4.00
4.50
ppm (t1)
Figure 7. TOCSY spectrum (D2O, 60 C) of BLPS fraction isolated from black liquor. Top
image represents the fragment of E-(1-3)-D-glucan backbone.
The chemical analysis of the sugars of TCF and ECF bleached pulps did not
show significant changes when compared to the unbleached pulp (Table 1). This
indicates that no specific removal of xylan from pulp took place during
bleaching. The chemical composition of the xylans isolated from TCF and ECF
bleached pulps was also similar to the xylan from unbleached pulp, although a
very small decrease in MeGlcpA content in pulp xylans after bleaching was
detected (Tables 2 and 3). This fact may be explained by a partial degradation
of MeGlcpA to HexA under alkaline conditions, which are inaccessible for the
analysis by methanolysis. This explanation was further supported by 1H NMR
analysis, that showed a relative increase of the HexA content and a decrease of
MeGlcpA moieties in the xylan from TCF bleached pulp (Figure 6). In contrast
to the xylan from TCF bleached pulp, the xylan from ECF bleached pulp did not
contain HexA residues, which were degraded upon bleaching with chlorine
dioxide (Figure 6). Taking into account that uronic moieties strongly affect the
papermaking properties (Lindstrm, 1992) and brightness stability (Buchert at
al. 1997) of cellulosic pulps this knowledge may be important to explain the
different response of pulps bleached employing TCF and ECF sequences.
234
5. Resultados y discusin
The xylans from bleached pulps (either TCF or ECF) did not show any acetyl
groups, as revealed by 1H NMR analysis. Hence, alkaline bleaching stages
favored the removal of residual acetyl groups from xylan of unbleached pulp.
Xylans from bleached pulps possessed slightly higher molecular weight (12 kDa
in TCF pulp and 14 kDa in ECF pulp), when compared to this in unbleached
pulp (Figure 2). This fact may be explained by a predominant removal of low
molecular weight xylan fractions structurally associated to residual lignin during
bleaching.
4. Conclusions
The structure of the heteroxylan isolated from sisal fibers has been characterized
and its behavior during soda/AQ pulping and TCF/ECF bleaching has been
studied. The data indicates that the heteroxylan backbone is composed by
(14)-linked -D-xylopyranosyl units (Xylp) partially ramified with terminal
(12)-linked 4-O-methyl--D-glucuronosyl (MeGlcpA, 9 %mol.) and a small
proportion of -D-glucuronosyl (GlcpA) residues. Around 61mol% of the Xylp
residues are acetylated, the major proportion of acetyl groups being attached at
the O-3 position of the Xylp residues (39 %mol.), followed by acetylation at the
O-2 position (13 %mol.) and diacetylation at both O-2 and O-3 positions
(9%mol.). The molecular weight (Mw) of the heteroxylan was of 18 kDa.
Around 40% of xylan was removed during soda pulping. However, the major
polysaccharides found in the black liquor were E-glucans rather than xylans.
Sisal xylan suffered a significant depolymerisation (Mw decreased to almost
half) and deacetylation (95%) during pulping. Terminal MeGlcpA residues were
partially removed (to about 30%) or converted to HexA in a large extent. HexA
revealed to be relatively stable during TCF bleaching with hydrogen peroxide
and were predominant among uronic moieties of xylan. Since all HexA were
degraded during ECF bleaching with chlorine dioxide, the final pulp contained a
xylan with rather small amount of uronosyls (MeGlcpA residues). A small
proportion of MeGlcpA residues (15% from initial amounts), remaining intact
during soda pulping, were additionally converted to HexA residues during alkali
bleaching stages. After bleaching, the residual acetyl groups were completely
removed from the pulp xylan. It was suggested that a low molecular weight
fraction of xylan, probably associated to residual lignin, was removed from upon
bleaching.
Acknowledgements
This study has been supported by the Spanish Projects AGL2005-01748 and
AGL2008-00709 and the EU BIORENEW project (NMP2-CT-2006-26456).
We thank CELESA (Tortosa, Spain) for providing the samples. G.M. thanks the
Spanish Ministry of Education for a FPI fellowship
235
5. Resultados y discusin
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Vuorinen, T., Fagerstrm, P., Buchert, J., Tenkanen, M., & Teleman, A. (1999).
Selective hydrolysis of hexenuronic acid groups and its application in ECF
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Publicacin IX:
Marques G., Gamelas J. A., Ruiz-Dueas F.J., del Ro J.C., Evtuguin D.V.,
Martnez A.T. and Gutirrez A. (2010) Delignification of eucalypt kraft pulp
with manganese-substituted polyoxometalate assisted by fungal versatile
peroxidase. Bioresource Technology, 101, 5935-5940.
240
5. Resultados y discusin
Gisela Marques a, Jos A.F. Gamelas b,, Francisco J. Ruiz-Dueas c, Jos C. del Rio a, Dmitry
V. Evtuguin b, Angel T. Martnez c, Ana Gutirrez a
a
Instituto de Recursos Naturales y Agrobiologa de Sevilla, CSIC, PO Box 1052, E-41080
Seville, Spain
b
University of Aveiro, CICECO, 3810-193, Aveiro, Portugal
c
Centro de Investigaciones Biolgicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid
Abstract
Oxidation of the manganese-substituted polyoxometalate [SiW11MnII(H2O)O39]6-
(SiW11MnII) to [SiW11MnIII(H2O)O39]5-, one of the most selective
polyoxometalates for the kraft pulp delignification, by versatile peroxidase (VP)
was studied. First, SiW11MnII was demonstrated to be quickly oxidized by VP at
room temperature in the presence of H2O2 (Km= 6.40.7 mM and kcat= 472 s-1).
Second, the filtrate from eucalypt pulp delignification containing reduced
polyoxometalate was treated with VP/H2O2, and 95-100% reoxidation was
attained. In this way, it was possible to reuse the liquor from a first SiW11MnIII
stage for further delignification, in a sequence constituted by two
polyoxometalate stages, and a short intermediate step consisting of the addition
of VP/H2O2 to the filtrate for SiW11MnII reoxidation. When the first ClO2 stage
of a conventional bleaching sequence was substituted by the two-stage
delignification with polyoxometalate (assisted by VP) a 50% saving in ClO2 was
obtained for similar mechanical strength of the final pulp.
Keywords: Polyoxometalate, versatile peroxidase, oxidative delignification, pulp
bleaching, eucalypt kraft pulp
1. Introduction
The residual lignin remaining after wood pulping is the target of the bleaching
process to produce high quality pulp for the papermaking. This plant polymer,
which is responsible for the undesirable dark color and photoyellowing of pulp,
must be attacked with minimal polysaccharides damage to preserve the physical
properties of the bleached pulp.
In the middle of the 1990's, polyoxometalates (POM) were proposed as an
environmentally-friendly alternative to the chlorine-based bleaching reagents,
as well as to conventional alkaline oxygen pre-bleaching (Evtuguin and Neto,
1997; Weinstock et al., 1997). POM have been evaluated for the
bleaching/delignification of pulps both as reagents under anaerobic conditions
(in this case a second stage is required for POM reoxidation and reuse) or as
catalysts under aerobic conditions (Gamelas et al., 2008; Gaspar et al., 2007;
241
5. Resultados y discusin
Weinstock et al., 1997). Applied as catalysts, POM oxidizes the residual lignin
in pulp, and the reduced form of POM is reoxidized by molecular oxygen at the
same stage. Therefore, it is possible to reuse the POM solutions in a closed loop.
The thermodynamic conditions required for lignin oxidation and reoxidation of
the POM are related to the corresponding redox potentials as follows: E (Lignin)
< E (POM) < E (O2) = 1.22 0.059 pH.
Several POM types, mostly with the Keggin-type structure (Fig. 1), have
been considered for kraft pulp delignification, such as [SiW11VO40]5-,
[SiW10V2O40]6-, SiW10.1Mo1.0V0.9O40, and [SiW11Mn(H2O)O39]5- (SiW11Mn)
(Gamelas et al., 2005a; 2008; Gaspar et al., 2003; 2007; 2009; Weinstock et al.,
1997; 2001). However, some of them possessing high M(n+1)/n redox potentials
(E = +0.70.8 V), although lower than oxygen redox potential, are hardly
reoxidized even at extreme conditions of oxygen pressure and temperature
(Gamelas et al., 2005a; Gaspar et al., 2003; Weinstock et al., 1997), thus,
limiting their reuse for pulp delignification.
In particular, SiW11Mn has been found to be highly selective in pulp
delignification (Gamelas et al., 2005a; Gaspar et al., 2003). The SiW11Mn/O2
catalytic system has been compared to the conventional alkaline oxygen process
already used by the pulp industry. In addition to lignin removal, an important
advantage of the SiW11Mn-based process, when applied to eucalypt kraft pulps,
is the higher reduction of kappa number than in the alkaline oxygen process, due
to the degradation of hexenuronic acids at the low pH used in these reactions
(Gamelas et al., 2005a). However, SiW11MnII is very slowly reoxidized under
these conditions, limiting its practical application.
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5. Resultados y discusin
(Gamelas et al., 2005b; Tavares et al., 2004). Although laccase easily oxidized
VIV to VV in the former POM, the corresponding oxidation of MnII to MnIII in
the manganese-based POM was slow (less that 50% after 4 h at 45 C, and under
0.3 bar oxygen pressure) (Gamelas et al., 2005b). This urged the search for
alternative faster methods of oxidation of MnII-substituted POM.
In contrast to laccase, versatile peroxidase (VP) produced by fungi of the
genera Pleurotus and Bjerkandera is a high redox-potential enzyme able to
oxidize a variety of substrates, including free MnII, due to the presence of
different catalytic sites in its molecular architecture (Ruiz-Dueas et al., 2009).
VP is activated by H2O2 in a two-electron reaction yielding highly reactive
intermediate states. Activated VP can oxidize two substrate molecules in two
successive one-electron reactions. It has been demonstrated that MnIII, resulting
from MnII oxidation by VP or related manganese peroxidase (Ruiz-Dueas et
al., 2007), is stabilized in solution by the chelation of dicarboxylic acids of small
size produced by ligninolytic fungi. The manganic cation can, then, act as an
oxidizer of lignin contributing to wood delignification in nature (Wariishi et al.,
1992).
In the present work, reoxidation of the MnII-containing POM, SiW11MnII, by
the VP/H2O2 system, was studied for the first time. Based on the easy oxidation
of MnII (as a free ion or in POM complexes) by the enzyme a novel approach for
the delignification catalysis was developed. Reduced POM in the liquor from a
first eucalypt pulp delignification stage was reoxidized by VP, and the resultant
liquor mixed with the partially delignified pulp for a further delignification stage
in a simple POM-VP-POM trial. In addition, delignification of eucalypt pulp by
POM in a VP-assisted process was tested as a pre-bleaching stage to substitute
the first Cl2O stage in a conventional elemental chlorine free (ECF) bleaching
sequence.
243
5. Resultados y discusin
2002). The enzyme was activated in vitro after solubilization of inclusion bodies
in 8 M urea. The folding conditions included 0.15 M urea, 5 mM Ca2+, 20 M
hemin, a 4:1 oxidized-glutathione/reduced-glutathione ratio and 0.1 mg/mL
protein at pH 9.5. The active enzyme was purified in a single chromatographic
step (Resource-Q column, Pharmacia-Biotech) using a 00.3 M NaCl gradient
(2 mL/min) in 10 mM sodium tartrate (pH 5.5) containing 1 mM CaCl2. The
concentration of the enzyme was determined by spectrophotometry (H407 150
000 M-1 cm-1) (Ruiz-Dueas et al., 1999).
244
5. Resultados y discusin
liquor, and the solution was stirred at 20-25 C for 10 min. The liquor containing
the reoxidized POM (verified by visible absorption spectroscopy) was mixed
again with the filtered pulp and a second delignification stage was applied under
the same experimental conditions of the first stage. A two-stage experiment not
including the reoxidation step of POM by VP/H2O2 was also performed by
adding fresh POM (SiW11MnIII), acetate buffer and water to the washed pulp
obtained after the first stage.
After each delignification sequence the pulps were filtered and washed with
water until neutral filtrate. Alkaline extraction of pulps was carried out at 70 C
during 1 h and NaOH charge of 2% (on the dry pulp weight).
245
5. Resultados y discusin
40
30
k obs (s )
-1
20
K m (6.4 0.7) mM
k cat (47.3 2) s-1
10 Efficiency (7.36 0.6) mM-1 s -1
0
0 5 10 15 20 25 30
[SiW 11MnII] (mM)
Fig. 2. Michaelis-Menten kinetics of SiW11MnII oxidation by VP. The Km, kcat and efficiency
values (means and standard errors) are shown inside the plot.
The high affinity of VP for free MnII is due to the existence of a specific
catalytic site in this enzyme constituted by three acidic residues forming a small
channel on the internal heme propionate, where the free metal cation is oxidized
(Ruiz-Dueas et al., 2007). The lower efficiency observed for SiW11MnII
oxidation by VP was in the order of those reported both for veratryl alcohol
oxidation taking place at a tryptophan residue exposed to the solvent (Prez-
Boada et al., 2005), and for oxidation of phenols at the edge of the main heme
access channel (Ruiz-Dueas et al., 2008). This suggested that SiW11MnII could
be oxidized in one of these two easily accessible catalytic sites, and not in the
narrow channel described for free MnII that most probably provides a limited
access to the bulky SiW11MnII. A detailed kinetic study of different VP variants
mutated at the three catalytic sites mentioned above would be necessary to
definitively identify the SiW11MnII oxidation site in VP.
246
5. Resultados y discusin
In the assays performed with the SiW11MnII solution (Table 1A), the extent
of POM oxidation (for a fixed amount of enzyme) increased with the H2O2/POM
ratio until a 0.5-0.6 molar ratio, and then decreased at higher ratios. Using this
H2O2/POM ratio (0.5-0.6), 95-100% POM oxidation was obtained in less than 5
min, with a POM/VP ratio ~2200. These values were in agreement with the
stoichiometry of the global enzymatic reaction, which predicts that 0.5 moles of
H2O2 will be needed to oxidize 1 mole of SiW11MnII. For the H2O2/POM ratio of
0.8, only 57% oxidation of SiW11MnII was obtained, indicating enzyme
inactivation by the excess of H2O2 (Valderrama et al., 2002). If the amount of
enzyme was reduced to about 50%, keeping the H2O2/POM ratio of 0.5, the
oxidation extent also decreased (to only 55%) due to the increased H2O2/VP
ratio. However, when the later assay was carried out by adding the H2O2 in
several steps, without exceeding a 500-fold molar excess of H2O2 in each
addition, the extent of oxidation (94%) was similar to that attained using a
higher amount of enzyme. These data confirmed VP inactivation by H2O2 (even
247
5. Resultados y discusin
in the presence of enough amount of SiW11MnII to consume all the H2O2) and
showed that the enzyme dose can be reduced by stepwise addition of H2O2 (to
prevent VP inactivation). No POM oxidation was observed in the absence of
H2O2 or without enzyme.
In the assays with the liquor from POM delignification of eucalypt kraft pulp
(Table 1B), which were performed using a POM/VP ratio ~2200, and varying
the H2O2/POM ratio between 0 and 1, the highest POM oxidation degrees (over
90%) were obtained at the H2O2/POM ratios of 0.4-0.5. In the absence of H2O2,
some POM oxidation occurred suggesting that some substances present in the
delignification liquor may act as the enzyme oxidizing agents. In fact, for all the
assays performed with a H2O2/POM ratio up to 0.5, the oxidation of POM was
higher when the reactions were conducted in the delignification liquor than
those in the SiW11MnII aqueous solutions. Again, the use of H2O2/POM ratios t
0.6 did not improve POM reoxidation in the delignification liquor, and lower
rates were obtained. It was concluded that a H2O2/POM ratio around 0.5 and a
POM/VP ratio of 2000-3000 should be used to obtain near complete oxidation
of the manganese-substituted POM.
248
5. Resultados y discusin
1,5
Absorbance
b
0,5
a
0
300 400 500 600 700 800
Wavelength (nm)
Fig. 3. UV-vis spectra of the filtrate after 2-h treatment of eucalypt kraft pulp with POM/O2
(a), and after short incubation of this delignification liquor with VP and H2O2 at 20-25C (b),
revealing the typical SiW11MnII and SiW11MnIII spectra, respectively.
249
5. Resultados y discusin
Table 3. Cl2O consumption and oxidation equivalents (OXE) for eucalypt pulp
bleaching in a conventional ECF sequence and after substituting the first D-stage by
VP-assisted two-stage POM delignification (89% ISO final brightness)
D-Ep-D-Ep-D POM-VP-POMreoxE-D-Ep-D
a
ClO2 consumption 25 + 9 + 6 15 + 5
OXE b 90 134
a
As active chlorine in each D stage (kg/ton)
b
As moles of active chlorine per ton of dry pulp and kappa unit
4. Conclusions
In this work, we demonstrate that the reduced Mn-substituted POM, SiW11MnII,
can be oxidized by VP (in the presence of H2O2) following Michaelis-Menten
kinetics. This POM, whose oxidized form is highly selective for delignification,
was fully oxidized by VP/H2O2 at 20-25 C in less than 10 min. In this way, a
two-stage POM delignification process, including a fast intermediate step
consisting of the addition of VP (and H2O2) to the delignification filtrate for
POM reoxidation, was performed, resulting in 62% reduction of the pulp kappa
number and a viscosity loss of only 10%. The substitution of the first Cl2O stage
by a POM-VP-POMreox treatment in a conventional ECF bleaching sequence
allowed 50% Cl2O saving without decreasing the pulp strength properties.
250
5. Resultados y discusin
Table 4. Physical properties of unbeaten bleached pulps (89% ISO, and 65 g/m2) from a
conventional ECF sequence and after substituting the first Cl2O stage by VP-assisted two-stage
POM delignification
D-Ep-D-Ep-D POM-VP-POMreox-E-D-Ep-D
Beating degree (SR) 19 20
Bulk density (g/cm3) 0.56 0.57
Burst index (kPa.m2/g) 1.39 1.45
Tensile strength (N.m/g) 30.4 28.9
Tear index (mN.m2/g) 5.2 6.0
Elongation (%) 2.2 2.1
Stiffness (kN/m) 409 396
Opacity (%) 75.8 77.2
Internal bonds (Scott test, J/m2) 106 114
Air resistance (Gurley-100 mL, s) 0.8 0.8
Acknowledgements
This study has been supported by the EU project BIORENEW (NMP2-CT-
2006-026456) and the Spanish projects AGL2008-00709 and BIO2008-01533.
We thank ENCE (Pontevedra, Spain) for pulp samples. G. M. and F. J. R.-D.
thank the Spanish MICINN for a FPI fellowship and a Ramon y Cajal contract,
respectively.
References
Evtuguin, D.V., Neto, C.P., 1997. New polyoxometalate promoted method of
oxygen delignification. Holzforschung 51, 338-342.
Furtado, F.P., Evtuguin, D.V., Gomes, T.M., 2001. Effect of the acid stage in
ECF bleaching on Eucalyptus globulus kraft pulp bleachability and strength.
Pulp Paper Can. 102, 89-92.
Galli, C., Gentili, P., Pontes, A.S.N., Gamelas, J.A.F., Evtuguin, D.V., 2007.
Oxidation of phenols employing polyoxometalates as biomimetic models of
the activity of phenoloxidase enzymes. New J. Chem. 31, 1461-1467.
Gamelas, J.A.F., Evtuguin, D.V., Gaspar, A.R., 2008. Transition metal
complexes in the delignification catalysis. In: Varga, B., Kis, L. (Eds.),
Transition metal chemistry: New research. Nova Science Publishers, Inc.,
New York, pp. 15-57.
251
5. Resultados y discusin
Gamelas, J.A.F., Gaspar, A.R., Evtuguin, D.V., Neto, C.P., 2005a. Transition
metal substituted polyoxotungstates for the oxygen delignification of kraft
pulp. Appl. Cat. A:General 295, 134-141.
Gamelas, J.A.F., Pontes, A.S.N., Evtuguin, D.V., Xavier, A.M.R.B., Esculcas,
A.P., 2007. New polyoxometalate-laccase integrated system for kraft pulp
delignification. Biochem. Eng. J. 33, 141-147.
Gamelas, J.A.F., Tavares, A.P.M., Evtuguin, D.V., Xavier, A.M.B., 2005b.
Oxygen bleaching of kraft pulp with polyoxometalates and laccase applying a
novel multi-stage process. J. Mol. Catal. B Enzym. 33, 57-64.
Gaspar, A., Evtuguin, D.V., Neto, C.P., 2003. Oxygen bleaching of kraft pulp
catalysed by Mn(III)-substituted polyoxometalates. Appl. Cat. A:General
239, 157-168.
Gaspar, A.R., Gamelas, J.A.F., Evtuguin, D.V., Neto, C.P., 2007. Alternatives
for lignocellulosic pulp delignification using polyoxometalates and oxygen: a
review. Green Chem. 9, 717-730.
Gaspar, A.R., Gamelas, J.A.F., Evtuguin, D.V., Neto, C.P., 2009.
Polyoxometalate-catalyzed oxygen delignification process: kinetic studies,
delignification sequences and reuse of HPA-5-MnII aqueous solution. Chem.
Eng. Commun. 196, 801-811.
International Organisation for Standardization Documentation and Information,
2003. ISO Standards Collection on CD-ROM. Paper, board and pulps. ISO,
Geneva.
Prez-Boada, M., Doyle, W.A., Ruiz-Dueas, F.J., Martnez, M.J., Martnez,
A.T., Smith, A.T., 2002. Expression of Pleurotus eryngii versatile peroxidase
in Escherichia coli and optimisation of in vitro folding. Enzyme Microb.
Technol. 30, 518-524.
Prez-Boada, M., Ruiz-Dueas, F.J., Pogni, R., Basosi, R., Choinowski, T.,
Martnez, M.J., Piontek, K., Martnez, A.T., 2005. Versatile peroxidase
oxidation of high redox potential aromatic compounds: Site-directed
mutagenesis, spectroscopic and crystallographic investigations of three long-
range electron transfer pathways. J. Mol. Biol. 354, 385-402.
Ruiz-Dueas, F.J., Martnez, M.J., Martnez, A.T., 1999. Molecular
characterization of a novel peroxidase isolated from the ligninolytic fungus
Pleurotus eryngii. Mol. Microbiol. 31, 223-236.
Ruiz-Dueas, F.J., Morales, M., Garca, E., Miki, Y., Martnez, M.J., Martnez,
A.T., 2009. Substrate oxidation sites in versatile peroxidase and other
basidiomycete peroxidases. J. Exp. Bot. 60, 441-452.
252
5. Resultados y discusin
253
5. Resultados y discusin
Publicacin X:
Marques, G., Molina, S., Babot, E.D., Lund, H., del Ro, J.C. y Gutirrez, A.
Exploring the potential of a fungal manganese-containing for pitch control and
pulp delignification. Bioresource Technology (in preparation).
254
5. Resultados y discusin
Gisela Marquesa, Setefilla Molinaa, Esteban D. Babota, Henrik Lundb, Jos C. del Roa, Ana
Gutirreza
a
Instituto de Recursos Naturales y Agrobiologa de Sevilla, CSIC, PO Box 1052,
E-41080, Seville, Spain
b
Novozymes A/S, Krogshoejvej 36, DK-2880 Bagsvaerd, Denmark
Abstract
The potential of the lipoxygenase from Gaeumannomyces graminis to remove
lipophilic extractives from eucalypt and flax pulps is explored. Pulp treatments
with the lipoxygenase were performed in the presence and absence of linoleic
acid, and with and without a subsequent hydrogen peroxide stage. The highest
removal of lipophilic extractives from eucalypt pulp, including conjugated
sterols (about 40% removal), and free sterols (up to 10% removal), was attained
in the lipoxygenase treatment with linoleic acid followed by the peroxide stage.
Different degradation patterns were observed among the lipophilic compounds
in flax pulp with the lipoxygenase treatment, although a high removal (from
55% to 70%) of all extractives classes, including alkanes, fatty alcohols, and
free and glycosylated sterols, was attained in most cases. Reactions of the
lipoxygenase with model lipid mixtures were carried out to better understand the
degradation patterns observed in pulps. Pulp delignification by the lipoxygenase
treatments was also evaluated.
Keywords: Lipoxygenase, Gumannomyces graminis, Fungal enzymes, Paper
pulps, Pitch deposits, Lignin removal.
1. Introduction
The non-polar extractable fraction from wood and nonwood fibers, commonly
referred to as lipophilic extractives, includes fatty and resin acids, fatty alcohols,
alkanes, sterols, sterol esters and triglycerides. These lipophilic compounds are
the precursors of the so-called pitch deposits within the pulp and paper
manufacturing processes (Back and Allen 2000). Pitch deposition results in low
quality pulp, and can cause the shutdown of pulp mill operations. Specific issues
related to pulps with high extractives content include runnability problems, spots
and holes in the paper, and sheet breaks.
In addition to physicochemical methods, biological methods including both
enzymes and microorganisms (Gutirrez et al. 2001a; 2009), have been
investigated to control pitch problems in the pulp and paper industry. Lipases,
which hydrolyze triglycerides, are applied with success in softwood (mainly
pine) mechanical pulping at mill scale (Fujita et al. 1992) and find widespread
255
5. Resultados y discusin
use nowadays. However, pitch problems in most of the chemical and mechanical
processes using other raw materials have not yet been solved. Other compounds,
such as free and esterified sterols, resin acids, fatty alcohols and alkanes, are
responsible for pitch problems in these processes (del Ro et al. 1999; 2000;
Gutirrez and del Ro 2005b). In addition to lipases, the use of sterol esterases
has also been suggested (Barfoed 2000; Calero-Rueda et al. 2004; Kontkanen et
al. 2004) because sterol esters are often at the origin of pitch deposits owing to
their high tackiness and resistance to kraft cooking. However, free sterols
released by the action of these esterases are as problematic as sterol esters.
On the other hand, the modification of some lipophilic extractives by the use
of laccases has been reported (Buchert et al. 2002; Molina et al. 2008; Zhang et
al. 2000; 2005). In contrast to lipases and sterol esterases, laccases are oxidative
enzymes whose action is directed toward some aromatic compounds (such as
phenols and anilines), although their reactivity with some unsaturated lipids was
demonstrated. The interest on laccases as industrial biocatalysts has increased
after discovering the effect of some synthetic compounds (Bourbonnais and
Paice 1990; Call 1994) expanding the action of laccases to non-phenolic
aromatics and, therefore, increasing their potential in the degradation of lignin
and other aromatic compounds (Riva 2006; Rodrguez-Couto and Toca 2006;
Widsten and Kandelbauer 2008). Moreover, the use of laccases in the presence
of redox mediators has recently been described for the removal of the lipophilic
extractives responsible for pitch deposition from wood and nonwood paper
pulps (Gutirrez et al. 2006a; 2006b; 2007; Molina et al. 2008).
In addition to laccases, other oxidative enzymes, such as soybean
lipoxygenases have been tested for the degradation of extractives in softwood
thermo-mechanical pulp (Zhang et al. 2007). Earlier work had also suggested
the use of lipoxygenases to reduce model wood pitch mixtures in pulp and
paper (Borch et al. 2003). Lipoxygenases are non-heme iron-containing
dioxygenases which catalyze the oxidation of polyunsaturated fatty acids to
hydroperoxides. Despite extensive studies on the properties, genetic information
and physiological functions of this group of enzymes, there is a lack of
utilization of these natural abundant enzymes in industrial processing. The
specific activity of lipoxygenases to degrade linoleic acid leads to a potential
application in papermaking processes to degrade wood extractives that have
adverse effects on pulp and paper properties. In the present work, we study the
capability of the lipoxygenase from the fungus Gaeumannomyces graminis, to
remove lipophilic extractives from hardwood (eucalypt) and nonwoody (flax)
pulps. This enzyme is a manganese-containing lipoxygenase with several
exceptional features different from other lipoxygenases (Hamberg et al. 1998;
Su and Oliw 1998). It has a broad pH range (being active between pH 5 and 11),
and good heat stability (being active at temperatures up to 60C) (Su and Oliw
1998) which confer great potential of use under mill process conditions. Since
the oxidation of polyunsaturated fatty acids by lipoxygenase leads to the
256
5. Resultados y discusin
formation of reactive fatty acid hydroperoxides and lipid radicals (Prigge et al.
1997) that can co-oxidize lignin in addition to lipids (Kapich et al. 2010),
delignification of this pulp mediated by lipoxygenase treatment in the presence
of linoleic acid was also investigated.
2.3. Lipoxygenase
The lipoxygenase (GLOX) used was provided by Novozymes (Bagsvaerd,
Denmark) and obtained from the fungus G. graminis. GLOX activity was
130000 units per mg, where one unit will cause an absorbance increase at 234
nm of 0.001 units per min, at pH 7.0 and 30C, when linoleic acid is used as
substrate in a reaction volume of 1.0 ml (light path 1 cm).
257
5. Resultados y discusin
258
5. Resultados y discusin
259
5. Resultados y discusin
100% 8
(a)
Relative response
1 10
2+3 11
4
2 4 6 8 10 12 14 16 18 20
Retention time (min)
100% 7
(b)
Relative response
1 2+3 8
6
4
5
10
2 4 6 8 10 12 14 16 18 20
Retention time (min)
Figure 1. GC chromatograms of silylated lipid extract from eucalypt kraft (a), and flax soda
(b) pulps. Peak identification: 1, palmitic acid; 2, oleic acid; 3, linoleic acid; 4, stearic acid; 5,
nonacosane; 6, hexacosanol; 7, octacosanol; 8, sitosterol; 9, triacontanol; 10, sterol
glycosides; and 11, sterol esters
260
5. Resultados y discusin
261
5. Resultados y discusin
OH HO
a b
OH
d e
Figure 2. Chemical structures of the model compounds representative for main paper pulp
lipophilic extractives used in the enzymatic reactions: (a) linoleic acid; (b) sitosterol; (c)
cholesteryl linoleate; (d) nonacosane and (e) octacosanol.
262
5. Resultados y discusin
100% 1 3
(a)
Relative response
4 6 8 10 12 14 16
Retention time (minutes)
100%
(b)
3
Relative response
5
4 7
2 6
4 6 8 10 12 14 16
Retention time (minutes)
263
5. Resultados y discusin
HO O
HO OH
f g
O O
O H O
h i
Figure 4. Chemical structures of the main oxidized derivatives identified after the
lipoxygenase reactions with free sterols (sitosterol) and sterol esters (cholesteryl linoleate): (f)
7-ketositosterol; (g) 7E-hydroxysitosterol; (h) cholesta-3,5-dien-7-one and (i) cholesteryl 9-
oxononanoate.
264
5. Resultados y discusin
Table 3. Properties of eucalypt pulp treated with the G. graminis lipoxygenase (GLOX)
in the absence and presence of linoleic acid (LA) before and after a H2O2 stage (P), and
control without enzyme
Control GLOX GLOX/LA
initial P initial P initial P
Kappa number 13.5 10.7 12.8 9.5 12.9 9.5
Brightness (% ISO) 44.0 55.9 43.8 58.5 43.9 59.3
Intrinsic viscosity (mL/g) 1140 925 1148 800 1143 767
In the case of flax pulp (Table 4), an increase of 0.8 and 1.2 points of ISO
brightness was achieved after the enzymatic treatment followed by the hydrogen
peroxide stage, in the absence and presence of linoleic acid, respectively (no
increase of brightness was observed before the hydrogen peroxide stage). No
significant decrease of kappa number (lower than 1 point) was observed after the
enzymatic treatment of flax pulp (before the peroxide stage). After peroxide, the
kappa number did not decrease and the viscosity increased.
Table 4. Properties of flax pulp treated with the G. graminis lipoxygenase (GLOX) in
the absence and presence of linoleic acid (LA) before and after a H2O2 stage (P), and
control without enzyme
Control GLOX GLOX/LA
initial P initial P initial P
Kappa number 9.3 5.1 9.1 5.2 8.6 5.5
Brightness (% ISO) 35.1 61.2 35.1 62 35.3 62.4
Intrinsic viscosity (mL/g) 787 535 762 648 779 602
265
5. Resultados y discusin
2
100%
1 4
(a)
3
Relative response
4 6 8 10 12 14
Retention time (minutes)
100%
(b)
Relative response
2
4
4 6 8 10 12 14
Retention time (minutes)
4. Conclusions
The potential of the lipoxygenase from G. graminis to remove lipophilic
extractives from a hardwood (eucalypt) and a nonwood (flax) pulp has been
studied. A removal up to 40% of esterified and glycosylated sterols was
266
5. Resultados y discusin
Acknowledgements
This study has been supported by the Spanish projects BIO2007-28719-E and
AGL2008-00709 and the EU project BIORENEW (NMP2-CT-2006-026456).
Novozymes (Bagsvaerd, Denmark) is acknowledged for GLOX supply and
ENCE (Pontevedra, Spain) and CELESA (Tortosa, Spain) for eucalypt and flax
paper pulp samples, respectively. J. Romero (ENCE) and T. Vidal (UPC,
Barcelona, Spain) are acknowledged for pulp properties determination.
References
Back EL, Allen LH (2000) Pitch control, wood resin and deresination. TAPPI
Press, Atlanta
Barfoed M (2000) Methods of hydrolyzing cholesterol esters by using a
Pseudomonas fragi cholesterol esterase. Patent (International) WO9423052:
Borch K, Franks N, Lund H, Xu H, Luo J (2003) Oxidizing enzymes in the
manufacturing of paper materials. Patent (USA)US 2003-0124710 A1
Bourbonnais R, Paice MG (1990) Oxidation of non-phenolic substrates. An
expanded role for laccase in lignin biodegradation. FEBS Lett 267:99-102
Buchert J, Mustranta A, Tamminen T, Spetz P, Holmbom B (2002)
Modification of spruce lignans with Trametes hirsuta laccase. Holzforschung
56:579-584
Calero-Rueda O, Gutirrez A, del Ro JC, Prieto A, Plou FJ, Ballesteros A,
Martnez AT, Martnez MJ (2004) Hydrolysis of sterol esters by an esterase
from Ophiostoma piceae: Application for pitch control in pulping of
Eucalyptus globulus wood. Intern J Biotechnol 6:367-375
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5. Resultados y discusin
Call H-P (1994) Verfahren zur Vernderung, Abbau oder Bleichen von Lignin,
ligninhaltigen Materialien oder hnlichen Stoffen. Patent (International) WO
94/29510:
Camarero S, Garca O, Vidal T, Colom J, del Ro JC, Gutirrez A, Gras JM,
Monje R, Martnez MJ, Martnez AT (2004) Efficient bleaching of non-wood
high-quality paper pulp using laccase-mediator system. Enzyme Microb
Technol 35:113-120
del Ro JC, Gutirrez A, Gonzlez-Vila FJ (1999) Analysis of impurities
occurring in a totally-chlorine free-bleached Kraft pulp. J Chromatogr 830:227-
232
del Ro JC, Gutirrez A, Gonzlez-Vila FJ, Martn F, Romero J (1998)
Characterization of organic deposits produced in the Kraft pulping of
Eucalyptus globulus wood. J Chromatogr 823:457-465
del Ro JC, Romero J, Gutirrez A (2000) Analysis of pitch deposits produced in
Kraft pulp mills using a totally chlorine free bleaching sequence. J Chromatogr
A 874:235-245
Fujita Y, Awaji H, Taneda H, Matsukura M, Hata K, Shimoto H, Sharyo M,
Sakaguchi H, Gibson K (1992) Recent advances in enzymic pitch control.
Tappi J 75 (4):117-122
Gutirrez A, del Ro JC (2001) Gas chromatography-mass spectrometry
demonstration of steryl glycosides in eucalypt wood, kraft pulp and process
liquids. Rapid Commun Mass Spectrom 15:2515-2520
Gutirrez A, del Ro JC (2003a) Lipids from flax fibers and their fate in alkaline
pulping. J Agric Food Chem 51:4965-4971
Gutirrez A, del Ro JC (2003b) Lipids from flax fibers and their fate in alkaline
pulping (Vol 51, pg 4965, 2003). J Agric Food Chem 51:6911-6914
Gutirrez A, del Ro JC (2005a) Chemical characterization of pitch deposits
produced in the manufacturing of high-quality paper pulps from hemp fibers.
Bioresource Technol 96:1445-1450
Gutirrez A, del Ro JC (2005b) Pitch episodes produced during the
manufacturing of high-quality paper pulps from hemp fibers. Bioresour
Technol (submitted):
Gutirrez A, del Ro JC, Gonzlez-Vila FJ, Martn F (1998) Analysis of
lipophilic extractives from wood and pitch deposits by solid-phase extraction
and gas chromatography. J Chromatogr 823:449-455
Gutirrez A, del Ro JC, Gonzlez-Vila FJ, Martn F (1999) Chemical
composition of lipophilic extractives from Eucalyptus globulus Labill. wood.
Holzforschung 53:481-486
268
5. Resultados y discusin
269
5. Resultados y discusin
270
5. Resultados y discusin
271
6
CONCLUSIONES
273
6. Conclusiones
5. Los principales enlaces entre las unidades de lignina en todas las fibras
estudiadas son de tipo aril-ter -O-4. Tambin estn presentes unidades
de tipo resinol -, fenilcumarano -5/-O-4 y espirodienona -1/-O-
. La mayor proporcin de enlaces -O-4 se encuentra en las ligninas
del kenaf, sisal, abac y curau, las cuales al tener tambin mayor
proporcin de unidades S, son ms fcilmente deslignificables.
274
6. Conclusiones
275
7
ANEXOS
Anexo 1. Peso de pasta ideal (c/ 7,5% humedad) para la determinacin del
ndice kappa. Adaptado de un documento realizado por Armindo Gaspar de la
Universidad de Aveiro.
277
7. Anexos
f+ 0 1 2 3 4 5 6 7 8 9
30 0,958 0,960 0,962 0,964 0,966 0,968 0,970 0,973 0,975 0,997
40 0,979 0,981 0,983 0,985 0,987 0,989 0,991 0,994 0,996 0,998
50 1,000 1,002 1,004 1,006 1,099 1,011 1,013 1,015 1,017 1,019
60 1,002 1,024 1,026 1,028 1,030 1,033 1,035 1,037 1,039 1,042
70 1,004
278
7. Anexos
279
7. Anexos
0,00 0,01 0,02 0,03 0,04 0,05 0,06 0,07 0,08 0,09
1,0 0,000 0,010 0,020 0,030 0,040 0,049 0,059 0,069 0,078 0,088
1,1 0,097 0,107 0,116 0,125 0,134 0,144 0,153 0,162 1,171 0,180
1,2 0,189 0,198 0,207 0,216 0,224 0,233 0,242 0,250 0,259 0,268
1,3 0,276 0,285 0,293 0,302 0,310 0,318 0,326 0,335 0,343 0,351
1,4 0,359 0,367 0,375 0,383 0,391 0,399 0,407 0,415 0,423 0,431
1,5 0,438 0,446 0,454 0,462 0,469 0,477 0,484 0,492 0,499 0,507
1,6 0,514 0,522 0,529 0,537 0,544 0,551 0,558 0,566 0,573 0,580
1,7 0,587 0,594 0,601 0,608 0,615 0,622 0,629 0,636 0,643 0,650
1,8 0,657 0,664 0,671 0,678 0,684 0,691 0,698 0,705 0,711 0,718
1,9 0,725 0,731 0,738 0,744 0,751 0,757 0,764 0,770 0,777 0,783
2,0 0,790 0,796 0,802 0,809 0,815 0,821 0,827 0,834 0,840 0,846
2,1 0,852 0,858 0,865 0,871 0,877 0,883 0,889 0,895 0,901 0,907
2,2 0,913 0,919 0,925 0,931 0,937 0,943 0,949 0,954 0,960 0,966
2,3 0,972 0,978 0,983 0,989 0,995 1,001 1,006 1,012 1,018 1,023
2,4 1,029 1,035 1,040 1,046 1,051 1,057 1,062 1,068 1,073 1,079
2,5 1,084 1,090 1,095 1,101 1,106 1,111 1,117 1,122 1,127 1,133
2,6 1,138 1,143 1,149 1,154 1,159 1,164 1,170 1,175 1,180 1,185
2,7 1,190 1,196 1,201 1,206 1,211 1,216 1,221 1,226 1,231 1,236
2,8 1,241 1,246 1,251 1,256 1,261 1,266 1,271 1,276 1,281 1,286
2,9 1,291 1,296 1,301 1,306 1,310 1,316 1,320 1,325 1,330 1,335
3,0 1,339 1,344 1,349 1,354 1,358 1,363 1,368 1,373 1,377 1,382
3,1 1,387 1,391 1,396 1,401 1,405 1,410 1,414 1,419 1,424 1,428
3,2 1,433 1,437 1,442 1,446 1,451 1,455 1,460 1,464 1,469 1,473
3,3 1,478 1,482 1,487 1,491 1,496 1,500 1,504 1,509 1,513 1,517
3,4 1,522 1,526 1,531 1,535 1,539 1,544 1,548 1,552 1,556 1,561
3,5 1,565 1,569 1,573 1,578 1,582 1,586 1,590 1,595 1,599 1,603
3,6 1,607 1,611 1,615 1,620 1,624 1,628 1,632 1,636 1,640 1,644
3,7 1,648 1,653 1,657 1,661 1,665 1,669 1,673 1,677 1,681 1,685
3,8 1,689 1,693 1,697 1,701 1,705 1,709 1,713 1,717 1,721 1,725
3,9 1,729 1,732 1,736 1,740 1,744 1,748 1,752 1,756 1,760 1,764
4,0 1,767 1,771 1,775 1,779 1,783 1,787 1,790 1,794 1,798 1,802
4,1 1,806 1,809 1,813 1,817 1,821 1,824 1,828 1,832 1,836 1,839
4,2 1,843 1,847 1,851 1,854 1,858 1,862 1,865 1,869 1,873 1,876
4,3 1,880 1,884 1,887 1,891 1,894 1,898 1,902 1,905 1,909 1,912
4,4 1,916 1,920 1,923 1,927 1,930 1,934 1,937 1,941 1,944 1,948
280
7. Anexos
0,00 0,01 0,02 0,03 0,04 0,05 0,06 0,07 0,08 0,09
4,5 1,952 1,955 1,959 1,962 1,966 1,969 1,973 1,976 1,979 1,983
4,6 1,986 1,990 1,993 1,997 2,000 2,004 2,007 2,010 2,014 2,017
4,7 2,021 2,024 2,028 2,031 2,034 2,038 2,041 2,044 2,048 2,051
4,8 2,054 2,058 2,061 2,064 2,068 2,071 2,074 2,078 2,081 2,084
4,9 2,088 2,091 2,094 2,098 2,101 2,104 2,107 2,111 2,114 2,117
5,0 2,120 2,124 2,127 2,130 2,133 2,137 2,140 2,143 2,146 2,149
5,1 2,153 2,156 2,159 2,162 2,165 2,168 2,172 2,175 2,178 2,181
5,2 2,184 2,187 2,191 2,194 2,197 2,200 2,203 2,206 2,209 2,212
5,3 2,215 2,219 2,222 2,225 2,228 2,231 2,234 2,237 2,240 2,243
5,4 2,246 2,249 2,252 2,255 2,258 2,261 2,264 2,267 2,270 2,273
5,5 2,276 2,280 2,283 2,286 2,288 2,291 2,294 2,297 2,300 2,303
5,6 2,306 2,309 2,312 2,315 2,318 2,321 2,324 2,327 2,330 2,333
5,7 2,336 2,339 2,342 2,345 2,347 2,350 2,353 2,356 2,359 2,362
5,8 2,365 2,368 2,371 2,374 2,376 2,379 2,382 2,385 2,388 2,391
5,9 2,394 2,396 2,399 2,402 2,405 2,408 2,411 2,413 2,416 2,419
6,0 2,422 2,425 2,427 2,430 2,433 2,436 2,439 2,441 2,444 2,447
6,1 2,450 2,452 2,455 2,458 2,461 2,463 2,466 2,469 2,472 2,475
6,2 2,477 2,480 2,783 2,485 2,488 2,491 2,494 2,496 2,499 2,502
6,3 2,504 2,507 2,510 2,512 2,515 2,518 2,521 2,523 2,526 2,529
6,4 2,531 2,534 2,537 2,539 2,542 2,545 2,547 2,550 2,552 2,555
6,5 2,558 2,560 2,563 2,566 2,568 2,571 2,573 2,576 2,579 2,581
6,6 2,584 2,587 2,589 2,592 2,594 2,597 2,599 2,602 2,605 2,607
6,7 2,610 2,612 2,615 2,617 2,620 2,623 2,625 2,628 2,630 2,633
6,8 2,635 2,638 2,640 2,643 2,645 2,648 2,651 2,653 2,656 2,659
6,9 2,661 2,663 2,666 2,668 2,671 2,673 2,676 2,678 2,681 2,683
7,0 2,686 2,688 2,690 2,693 2,695 2,698 2,700 2,703 2,705 2,708
7,1 2,710 2,713 2,715 2,718 2,720 2,722 2,725 2,727 2,730 2,732
7,2 2,735 2,737 2,739 2,742 2,744 2,747 2,749 2,752 2,754 2,756
7,3 2,758 2,761 2,764 2,766 2,768 2,771 2,773 2,775 2,778 2,780
7,4 2,783 2,785 2,787 2,790 2,792 2,794 2,797 2,799 2,801 2,804
7,5 2,806 2,809 2,811 2,813 2,816 2,818 2,820 2,823 2,825 2,827
7,6 2,829 2,832 2,834 2,836 2,839 2,841 2,843 2,846 2,848 2,850
7,7 2,853 2,855 2,857 2,859 2,862 2,864 2,866 2,869 2,871 2,873
7,8 2,875 2,878 2,880 2,882 2,885 2,887 2,889 2,891 2,894 2,896
7,9 2,898 2,900 2,903 2,905 2,907 2,909 2,911 2,914 2,916 2,918
281
7. Anexos
0,00 0,01 0,02 0,03 0,04 0,05 0,06 0,07 0,08 0,09
8,0 2,920 2,923 2,295 2,927 2,929 2,932 2,934 2,936 2,938 2,940
8,1 2,943 2,945 2,947 2,949 2,951 2,954 2,956 2,958 2,960 2,962
8,2 2,964 2,967 2,969 2,971 2,973 2,975 2,978 2,980 2,982 2,984
8,3 2,986 2,988 2,991 2,993 2,995 2,997 2,999 3,001 3,003 3,006
8,4 3,008 3,010 3,012 3,014 3,016 3,018 3,020 3,023 3,025 3,027
8,5 3,029 3,031 3,033 3,035 3,037 3,040 3,042 3,044 3,046 3,048
8,6 3,050 3,052 3,054 3,056 3,058 3,061 3,063 3,065 3,067 3,069
8,7 3,071 3,073 3,075 3,077 3,079 3,081 3,083 3,085 3,087 3,090
8,8 3,092 3,094 3,096 3,098 3,100 3,102 3,104 3,106 3,108 3,110
8,9 3,112 3,114 3,116 3,118 3,120 3,122 3,124 3,126 3,128 3,130
9,0 3,132 3,134 3,136 3,138 3,340 3,142 3,144 3,147 3,149 3,151
9,1 3,153 3,155 3,157 3,159 3,161 3,163 3,165 3,166 3,168 3,170
9,2 3,172 3,174 3,176 3,178 3,180 3,182 3,184 3,186 3,188 3,190
9,3 3,192 3,194 3,196 3,198 3,200 3,202 3,204 3,206 3,208 3,210
9,4 3,212 3,214 3,216 3,218 3,220 3,222 3,223 3,225 3,227 3,229
9,5 3,231 3,233 3,265 3,237 3,239 3,241 3,242 3,245 3,247 3,249
9,6 3,250 3,252 3,254 3,256 3,258 3,260 3,262 3,264 3,266 3,268
9,7 3,270 3,271 3,273 3,175 3,277 3,273 3,281 3,283 3,285 3,887
9,8 3,288 3,290 3,292 3,294 3,296 3,298 3,300 3,302 3,303 3,305
9,9 3,307 3,309 3,311 3,313 3,315 3,316 3,318 3,320 3,322 3,324
10 3,326 3,344 3,363 3,381 3,399 3,416 3,434 4,452 3,469 3,487
11 3,504 3,521 3,538 3,554 3,571 3,588 3,604 3,620 3,636 3,653
12 3,669 3,684 3,700 3,716 3,731 3,747 3,762 3,777 3,792 3,807
13 3,822 3,837 3,852 3,866 3,881 3,895 3,910 3,924 3,938 3,952
14 3,966 3,980 3,994 4,008 4,021 4,035 4,048 4,062 4,075 4,088
15 4,101 4,115 4,128 4,141 4,153 4,166 4,179 4,192 4,204 4,217
16 4,229 4,242 4,254 4,266 4,279 4,291 4,303 4,315 4,327 4,339
17 4,351 4,362 4,374 4,386 4,397 4,409 4,420 4,432 4,443 4,455
18 4,466 4,477 4,488 4,499 4,510 4,521 4,532 4,543 4,554 4,565
19 4,576 4,586 4,597 4,608 4,618 4,629 4,639 4,650 4,660 4,670
282