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Valorización de diferentes cultivos lignocelulósicos para lafabricación de pasta de papel: Caracterización química,modificación estructural de sus constituyentes orgánicosdurante los procesos de cocción y blanqueo y aplicacionesbiotecnológicasMemoria que presentaGisela Marques Silvapara optar al título de Doctor en CienciasQuímicas por la Universidad de Sevilla.Sevilla, a 13 de Abril de 2010.

Valorización de diferentes cultivos lignocelulósicos para lafabricación de pasta de papel: Caracterización química,modificación estructural de sus constituyentes orgánicosdurante los procesos de cocción y blanqueo y aplicacionesbiotecnológicasVisado en Sevilla, a 13 de Abril de 2010LOS DIRECTORESDr. D. José C. del Río AndradeInvestigador Científico del CSICIRNAS-CSICDra. Dña. Ana Gutiérrez SuárezInvestigador Científico del CSICIRNAS-CSICEL TUTORDr. D. Alfonso Guiraúm PérezCatedrático de la Universidad de SevillaMemoria que presentaGisela Marques Silvapara optar al grado de Doctor en CienciasQuímicas por la Universidad de Sevilla.

DOCTOR D. LUIS CLEMENTE SALAS, DIRECTOR DEL INSTITUTO DERECURSOS NATURALES Y AGROBIOLOGÍA DE SEVILLA DELCONSEJO SUPERIOR DE INVESTIGACIONES CIENTÍFICASCERTIFICA: Que la presente Memoria de Investigación titulada “Valorizaciónde diferentes cultivos lignocelulósicos para la fabricación de pasta de papel:Caracterización química, modificación estructural de sus constituyentesorgánicos durante los procesos de cocción y blanqueo y aplicacionesbiotecnológicas”, presentada por Gisela Marques Silva para optar al grado deDoctor en Ciencias Químicas, ha sido realizada en el Departamento deBiotecnología Vegetal, bajo la dirección de los Drs. D. José C. del Río Andradey Dña. Ana Gutiérrez Suárez, reuniendo todas las condiciones exigidas a lostrabajos de Tesis Doctorales.En Sevilla, a 13 de Abril de 2010

AGRADECIMIENTOSEste trabajo se ha llevado a cabo en el Instituto de Recursos Naturales yAgrobiología de Sevilla (IRNAS-CSIC). Ha sido financiado por una beca I3P depostgrado del CSIC, por una beca FPI del Ministerio de Educación y Ciencia, por losproyectos nacionales de investigación AGL2005-01748 y AGL2008-00709, y por elproyecto europeo NMP2-CT-2006-026456.Quiero expresar mi sincero agradecimiento a las personas que tanto directa comoindirectamente han hecho posible la realización de esta Tesis:A los Dres. José Carlos del Río y Ana Gutiérrez, directores de esta Tesis, por todolo que me han aportado tanto a nivel científico, por sus conocimientos y enseñanzas,como a nivel personal, por la confianza en su trato personal y por sus consejos, y porestar siempre que los he necesitado. Por su esfuerzo y dedicación en esta Tesis.Al Prof. Ángel T. Martínez, del Centro de Investigaciones Biológicas (CIB-CSIC,Madrid), por ofrecerme la posibilidad de realizar en el CIB algunas estancias breves,aportándome numerosos y valiosos conocimientos, y por haber seguido el desarrollode esta Tesis.Al Prof. Dmitry Evtuguin, de la Universidad de Aveiro (Aveiro, Portugal), porofrecerme la posibilidad de realizar dos estancias en su grupo de investigación, poracogerme como un miembro más de su familia y por aportarme valiososconocimientos. Por su apoyo y consejos, y su excelente trato personal. También quieroagradecer al Dr. José Antonio Gamelas y a la Dra. Paula Pinto por su apoyo durantelas estancias en la Universidad de Aveiro.Al Prof. Alfonso Guiraúm, Catedrático de la Universidad de Sevilla, tutor de estaTesis, por toda su ayuda en la parte burocrática.A mi compañero de laboratorio durante los primeros años de la Tesis y amigo, elDr. Jorge Rencoret, por ayudarme siempre que lo he necesitado tanto en ellaboratorio como fuera, por su alegría, dándole siempre una vida muy suya allaboratorio sin dejar de lado la profesionalidad.A mis compañeras la Dra. Isabel María Rodríguez y Setefilla Molina, con las quehe coincidido en el inicio y mediados de esta Tesis, y a mis compañeros PepijnPrinsen, Alejandro Rico y Esteban Babot con los que he coincidido en el final de estaTesis. Un agradecimiento muy especial a Esteban por su apoyo y los buenosmomentos compartidos.A Dña. Trinidad Verdejo por hacer las pirólisis de mis numerosas muestras.

Al Prof. Jesús Jiménez-Barbero, al Dr. Iñaki Santos y a Lidia Nieto del CIB-CSIC, por sus múltiples análisis de NMR.A Gerardo Artal (CELESA) por suministrarme las muestras de diversas fibras y suspastas, al Dr. Javier Romero (ENCE) por las pastas de eucalipto y al Dr. Manuel J.Díaz Blanco (Universidad de Huelva) por las muestras de caña común y tagasaste.Al Dr. García Hortal (UPC, Terrassa) por las imágenes proporcionadas de lasfibras elementales de las muestras de lino, cáñamo, kenaf, yute, sisal y abacá que semuestran en la sección de Material y Métodos de esta Tesis.A mis compañeros del IRNAS, Rocío, Mari Trini, Agüi, Alegría, María Fernanda,Fátima, José María, Antonio y Jaime, que me acompañaron durante el inicio de estaTesis, y en particular a Fátima Sopeña que también me acompañó durante casi todala Tesis brindándome muy buenos momentos y consejos y por estar siempre allíincluso durante su post-doc en el extranjero.A los compañeros del CIB, Mario, Yuta, María, Ángeles, Miguel, Elvira, Davinia,Vero, Helena, Aitor, Eva y Beatriz, y en particular a Celia Méndez por su apoyo enmi primera estancia en el CIB y por los buenos momentos brindados. Quieroagradecer también al Dr. Javier Ruiz-Dueñas por su apoyo en las estanciasrealizadas en el CIB.A María Jesús Ortega, madre de Aitor del CIB, aunque sólo la conozco porInternet, por sus e-mails y por proporcionarme una de las fotos que se muestra en lasección de Introducción de esta Tesis.Y por último a mi hermana, a mi madre y a Augusto por el apoyo brindadoaunque estén lejos. A Dani por su paciencia, por escucharme hablar de experimentosque le son completamente ajenos y por estar ahí cuando más lo necesito, así como asu familia de Sevilla que desde que los conozco me han brindado todo su apoyo.Gracias a todos y gracias también, tan sólo por su existencia, a una nuevapersonita subacuática que ahora llevo dentro…

ABREVIATURASAQABTSBSTFA C HCEDCOSYDBODCMDMACDMSODTT2D-NMR3D-NMRECFFAOFIDGGCGC/MSHHBTHPLCHSQCAntraquinona2,2’-azinobis(3-etilbenzotiazolin-6-sulfonato)N,O-bis-(trimetilsilil)-trifluoroacetamidaDesplazamiento químico del carbonoDesplazamiento químico del protónCobre (II)-etilendiaminaEspectroscopia de correlación (“Correlation Spectroscopy”)Demanda biológica de oxígenoDiclorometanoN,N-dimetilacetamidaDimetilsulfóxidoDitiotreitolEspectroscopía de Resonancia Magnética Nuclear bidimensionalEspectroscopía de Resonancia Magnética Nuclear tridimensionalSecuencia de blanqueo libre de cloro elemental (“elementalchlorine free”)Organización de las Naciones Unidas para la Agricultura yAlimentación (“The Food and Agriculture Organization of theUnited Nations”)Detector de ionización de llama (“flame ionization detector”)Unidad guayacilpropano (o guayacilo)Cromatografía de gases (“gas chromatography”)Cromatografía de gases/espectrometría de masas (“gaschromatography/mass spectrometry”)Unidad 4-hidroxifenilpropano (o 4-hidroxifenilo)1-HidroxibenzotriazolCromatografía líquida de alta resoluciónEspectroscopía 2D de correlación heteronuclear de cuanto simple(“heteronuclear single-quantum correlation”)

HexAICP-OESIDIKIPTGISOITDLignina oxLiPMwMnPMWLNMROPCAPOMPOM 1--POM 2-POM oxPOM redPoPppbppmPy-GC/MSQrpmÁcidos hexenurónicosEspectrometría de emisión óptica con plasma acopladoinductivamente (“inductively coupled plasma-optical emissionspectrometry”)Diámetro interno (“internal diameter”)Índice KappaIsopropil tio--D-galactopiranósidoOrganización Internacional para la Estandarización,Documentación e Información (“International Organization forStandardization”)Detector de trampa de iones (“ion trap detector”)Productos de degradación oxidativa de la ligninaLignina peroxidasaMasa molecular (“molecular weight”)Manganeso peroxidasaLignina de madera molida (“milled wood lignin”)Espectroscopía de Resonancia Magnética Nuclear (“nuclearmagnetic resonance”)Etapa de deslignificación con oxígeno (en secuencia de blanqueo)Ácido p-cumáricopolioxometalatoPrimera etapa POM en los ensayos de deslignificaciónSegunda etapa POM en los ensayos de deslignificaciónPolioxometalato oxidadoPolioxometalato reducidoDoble etapa de blanqueo con peróxido de hidrógeno, la primerabajo oxígeno presurizadoPartes por billónPartes por millónPirólisis acoplada a cromatografía de gases/espectrometría demasas (“pyrolysis-gas chromatography/mass spectrometry”)Etapa de quelato (en secuencia de blanqueo)Revoluciones por minuto

SSPETAPPITCFTMAHTMPTMSDTMSTOCSYUUV/VISVPUnidad siringilpropano (o siringilo)Extracción en fase sólida (“solid phase extraction”)Technical Association of the Pulp and Paper IndustrySecuencia de blanqueo totalmente libre de cloro (“totally chlorinefree”)Hidróxido de tetrametilamonioPasta termomecánica (“thermomechanical pulp”)TrimetilsilildiazometanoTrimetilsililoEspectroscopia de correlación total (“Total CorrelationSpectroscopy”)Unidad de actividad enzimáticaEspectroscopía de ultravioleta/visiblePeroxidasa versátilCoeficiente de extinción molar

ÍNDICERESUMEN ..........................................................................................................11. INTRODUCCIÓN.........................................................................................51.1. CULTIVOS LIGNOCELULÓSICOS .....................................................51.1.1. Fibras procedentes de cultivos madereros ......................................51.1.2. Fibras procedentes de cultivos agrícolas ........................................71.2. ESTRUCTURA Y COMPOSICIÓN QUÍMICA DE LOSMATERIALES LIGNOCELULÓSICOS................................................91.2.1. Celulosa.........................................................................................101.2.2. Hemicelulosas...............................................................................111.2.3. Lignina ..........................................................................................131.2.4. Componentes de bajo peso molecular ..........................................201.3. UTILIZACIÓN DE CULTIVOS LIGNOCELULÓSICOS:PRODUCCIÓN DE PASTA DE CELULOSA .....................................231.3.1. Procesos de pasteado ....................................................................24Procesos mecánicos ...................................................................24Procesos químicos .....................................................................241.3.2. Procesos de blanqueo....................................................................261.4. PROBLEMÁTICA PLANTEADA POR LA PRESENCIA DELIGNINA Y LÍPIDOS EN LA PRODUCCIÓN DE PASTA DECELULOSA...........................................................................................281.5. BIOTECNOLOGÍA EN EL SECTOR DE LA PASTA DECELULOSA...........................................................................................291.5.1. Degradación enzimática de la lignina...........................................301.5.2. Degradación enzimática de lípidos: Control del pitch .................332. OBJETIVOS ................................................................................................373. MATERIAL Y MÉTODOS .......................................................................413.1. MATERIALES ......................................................................................413.1.1. Cultivos lignocelulósicos..............................................................41Lino ...........................................................................................41

Cáñamo .....................................................................................42Kenaf.........................................................................................43Yute...........................................................................................44Sisal...........................................................................................45Abacá ........................................................................................46Curauá .......................................................................................47Caña común...............................................................................47Tagasaste...................................................................................473.1.2. Pastas de papel ..............................................................................48Pastas de fibras no madereras ...................................................48Pastas de fibras madereras ........................................................483.1.3. Enzimas y mediadores ..................................................................49Lipoxigenasas ...........................................................................49Peroxidasas................................................................................49Polioxometalatos.......................................................................503.2. MÉTODOS ANALÍTICOS ...................................................................513.2.1. Aislamiento y análisis de los compuestos lipofílicos de lasfibras y pastas............................................................................51Fraccionamiento de los compuestos extraíbleslipofílicos mediante SPE...........................................................51Métodos de derivatización de los compuestos extraíbleslipofílicos ..................................................................................53Análisis de los extractos lipofílicos mediante GC y GC/MS........533.2.2. Aislamiento y análisis de la lignina de las fibras y pastas............54Determinación del contenido en lignina ...................................54Aislamiento de la lignina de las fibras......................................55Análisis de la lignina mediante Py-GC/MS..............................55Análisis de la lignina mediante DFRC .....................................56Análisis de la lignina mediante 2D-NMR ................................593.2.3. Aislamiento y análisis de las hemicelulosas de las fibras ypastas.........................................................................................62Preparación de la holocelulosa y aislamiento de losxilanos ......................................................................................62Análisis de azúcares neutros tras hidrólisis ácida....................62

Análisis de azúcares neutros y ácidos urónicos trasmetanolisis ácida ......................................................................63Determinación del peso molecular de los xilanosmediante SEC............................................................................63Análisis de la estructura de los xilanos mediante NMR ..........64Determinación del contenido en ácidos hexenurónicos ...........643.2.4. Otros análisis.................................................................................65Determinación de la fracción hidrosoluble de las fibras..........65Determinación del contenido en cenizas de las fibras..............65Análisis de metales y otros elementos en las fibras .................663.2.5. Tratamientos enzimáticos de las pastas ........................................66Tratamientos con lipoxigenasas................................................66Tratamientos con POM y peroxidasa versátil...........................67Determinación de las propiedades de las pastas .......................69Determinación de la blancura ISO........................................69Determinación del índice kappa ...........................................69Determinación de la viscosidad intrínseca ...........................70Determinación del contenido en ácidos hexenurónicos .......724. REFERENCIAS ..........................................................................................755. RESULTADOS Y DISCUSIÓN.................................................................91Publicación I: Marques G., Rencoret J., Gutiérrez A., del Río J.C.(2010) Evaluation of the chemical composition of different nonwoodyplant fibers used for pulp and paper manufacturing. TheOpen Agriculture Journal (in press)......................................................93Publicación II: del Río J.C., Marques G., Rencoret J., MartínezA.T. and Gutiérrez A. (2007) Occurence of naturally acetylatedlignin units. Journal of Agricultural and Food Chemistry, 55,5461-5468. ...........................................................................................111Publicación III: del Río J.C., Rencoret J., Marques G., GutiérrezA., Ibarra D., Santos J.I., Jiménez-Barbero J., Zhang L. andMartínez 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.............127Publicación IV: Marques G., Gutiérrez A. and del Río J.C. (2007)Chemical characterization of lignin and lipophilic fractions from

leaf fibers of curaua (Ananas erectifolius). Journal ofAgriculture and Food Chemistry, 55, 1327-1336................................151Publicación V: Coelho D., Marques G., Gutiérrez A., SilvestreA.R.D. and del Río J.C. (2007) Chemical characterization of thelipophilic fraction of Giant reed (Arundo donax) fibers used forpulp and paper manufacturing. Industrial Crops and Products,26, 229-236.. ........................................................................................173Publicación VI: Marques G., Gutiérrez A. and del Río J.C. (2008)Chemical composition of lignin and lipids from tagasaste(Chamaecytisus proliferus spp. palmensis). Industrial Cropsand Products, 28, 29-36......................................................................187Publicación VII: Marques G., del Río J.C. and Gutiérrez A. (2010)Lipophilic extractives from several nonwoody lignocellulosiccrops (flax, hemp, sisal, abaca) and their fate during alkalinepulping and TCF/ECF bleaching. Bioresource Technology, 101,260-267.................................................................................................203Publicación VIII: Marques G., Gutiérrez A., del Río J.C. andEvtuguin D.V. (2010) Acetylated heteroxylan from Agavesisalana and its behavior in alkaline pulping and TCF/ECFbleaching. Carbohydrate Polymers, (doi: 10-1016/j.carbpol.2010.02.043)................................................................221Publicación IX: Marques G., Gamelas J.A., Ruiz-Dueñas F.J., delRío J.C., Evtuguin D.V., Martínez A.T. and Gutiérrez A. (2010)Delignification of eucalypt kraft pulp with manganesesubstitutedpolyoxometalate assisted by fungal versatileperoxidase. Bioresource Technology, 101, 5935-5940. .....................241Publicación X: Marques G., Molina S., Babot E.D., Lund H., delRío J.C. and Gutiérrez A. Exploring the potential of a fungalmanganese lipoxygenase to remove lipophilic extractives frompaper pulps. Bioresource Technology (in preparation).......................2556. CONCLUSIONES.....................................................................................2737. ANEXOS ....................................................................................................277

RESUMENLa presente Tesis plantea el estudio de la composición química de losprincipales constituyentes de diferentes cultivos lignocelulósicos utilizadoscomo materia prima para la fabricación de pastas de celulosa de alta calidad,poniendo especial énfasis en la composición de la fracción lipofílica(responsable de la formación de los denominados depósitos de pitch) y de lalignina (cuya composición y estructura influyen decisivamente en el proceso dedeslignificación), así como en la composición de las hemicelulosas. Entre losmateriales estudiados se incluyen fibras no madereras del tallo de variasangiospermas dicotiledóneas, tales como lino (Linum usitatissimum), kenaf(Hibiscus cannabinus), cáñamo (Cannabis sativa) y yute (Corchoruscapsularis), así como fibras procedentes de hojas de angiospermasmonocotiledóneas como sisal (Agave sisalana), abacá (Musa textilis) y curauá(Ananas erectifolius). Otras fibras estudiadas fueron las procedentes de la cañacomún (Arundo donax) y de podas de árboles de tagasaste (Chamaecytisusproliferus spp. palmensis). Se estudió también la evolución de los constituyentesde los materiales lignocelulósicos durante la producción de pasta de papel. Paraello, se seleccionaron diversas pastas de celulosa a lo largo de los procesos depasteado (cocción sosa-antraquinona) y de blanqueo (procesos TCF y ECF).Finalmente, se ensayaron dos procedimientos biotecnológicos basados en lautilización de enzimas fúngicas para la eliminación tanto de lignina como delípidos residuales en pastas de celulosa.Los resultados obtenidos muestran que las diferentes materias primasestudiadas se caracterizan, en general, por un alto contenido en polisacáridos yun bajo contenido en lípidos y lignina, lo que las hace, en principio, favorablespara la producción de pasta de celulosa. Los compuestos lipofílicos presentes enlas fibras, analizados por GC y GC/MS, incluyen principalmente ácidos grasos,hidroxiácidos, alcoholes, ceras, alcanos y esteroles libres y conjugados (enforma de ésteres y glicósidos), entre otros. Los análisis indican que el contenidoy composición de las diferentes clases de lípidos varía considerablemente entrelas distintas fibras. Además, las diferentes clases de lípidos muestran distintocomportamiento durante los procesos de cocción y blanqueo. Así, las ceras sehidrolizan durante la cocción alcalina mientras que los ácidos grasos sedisuelven. Por el contrario, los alcanos, alcoholes grasos, esteroles ytriterpenoles, hidrocarburos esteroidales, cetonas y glicósidos de esteroles tienenbaja solubilidad en agua y son difíciles de eliminar de la pasta, por lo quesobreviven a la cocción. Se observó que entre los compuestos que sobreviven ala cocción, los esteroles libres se eliminan durante el blanqueo ECF pero resistenal blanqueo TCF, mientras que los glicósidos de esteroles se eliminan tanto en elblanqueo TCF como ECF. Finalmente, mientras que los ácidos grasos1

insaturados se eliminan durante los procesos de blanqueo ECF y TCF, los ácidosgrasos saturados, así como los alcanos y alcoholes grasos sobreviven a estassecuencias de blanqueo.En cuanto a las ligninas, su estructura y composición se estudió tanto pormétodos degradativos (pirólisis-GC/MS y DFRC) como espectroscópicos (2D-NMR). Los análisis mostraron un predominio de unidades de tipo siringilo (S)en el caso de las fibras liberianas de kenaf y yute, así como en todas las fibras dehojas (sisal, abacá y curauá). Por el contrario, las fibras de cáñamo, lino y cañacomún mostraron un predominio de unidades de tipo guayacilo (G). Esto fueespecialmente evidente en las ligninas de lino y cáñamo, con una relación S/Gde 0,1. La mayor relación S/G de las ligninas de kenaf, yute, sisal y abacá haceque estas fibras sean más fáciles de deslignificar a causa del menor grado decondensación de la lignina, a pesar de tener un mayor contenido en lignina. Losprincipales enlaces entre las unidades de lignina fueron de tipo aril-éter -O-4en todas las fibras estudiadas. También se observaron enlaces condensados -5/-O-4 (fenilcumarano), - (resinol) y -1/-O- (espirodienona). Lamayor proporción de enlaces no condensados -O-4 se encontró en las ligninasde kenaf, sisal, abacá y curauá, las cuales al tener también mayor proporción deunidades S son más fácilmente deslignificables. Por otro lado, en las ligninas dekenaf, sisal, abacá y curauá se encontraron unidades aciladas (con acetatos y/op-cumaratos) en el carbono de la cadena lateral y predominantemente sobreunidades S. Se demostró que la acilación tiene lugar a nivel de monómero y queel sinapil acetato, y otros monómeros acilados, se comportan como auténticosmonómeros de la lignina. Se demostró también, que el nivel de acilación de lalignina estaba relacionado con un alto contenido en unidades S y enlaces -O-4,así como con un menor contenido en enlaces -.También se estudió la composición química de las hemicelulosas y lasmodificaciones de las mismas durante los procesos de pasteado y blanqueo. Elestudio de las hemicelulosas tiene importancia debido a que los polisacáridos sedisuelven y/o degradan parcialmente durante el pasteado, lo que afecta alrendimiento del proceso y a la calidad de las pastas de celulosa. Los resultadosmostraron que las hemicelulosas de las fibras liberianas presentan una mayorvariabilidad en cuanto a su composición en azúcares neutros que las fibrasprocedentes de hojas. Así, mientras que en las fibras de lino y cáñamopredominan la manosa y la galactosa, en el kenaf y yute el monosacáridopredominante es la xilosa. Por otro lado, en todas las fibras de hojas estudiadas(sisal, abacá y curauá) se observó un predominio de la xilosa. Un estudio enprofundidad de la estructura de las hemicelulosas de sisal mostró que estánconstituidas fundamentalmente por un glucuronoxilano acetilado cuya cadenaprincipal está formada por unidades de -D-xilopiranosa parcialmenteramificada con residuos glucuronosilos. Esta hemicelulosa sufre una2

despolimerización y desacetilación significativa durante el proceso de pasteado.Los grupos acetilo residuales que quedaban en la pasta cruda se eliminaroncompletamente tras el blanqueo.Finalmente, se estudiaron dos procedimientos biotecnológicos basados en eluso de enzimas fúngicas para la eliminación de la lignina residual de pastas asícomo de los compuestos extraíbles lipofílicos responsables de la formación dedepósitos de pitch durante el proceso de fabricación de pasta de papel. Estosprocedimientos incluyeron la utilización de un sistema compuesto de unpolioxometalato y una enzima de tipo peroxidasa producida por el hongoPleurotus eryngii, y una lipoxigenasa producida por el hongo Gaeumannomycesgraminis. Los resultados obtenidos mostraron la eficacia del sistemapolioxometalato-peroxidasa para eliminar la lignina residual de la pasta y de lalipoxigenasa para degradar parte de los compuestos lipofílicos responsables dela formación de los depósitos de pitch.La presente Tesis incluye los siguientes apartados: i) una introducción generalsobre los cultivos lignocelulósicos, su interés industrial, su estructura ycomposición, y los procesos utilizados para la producción de pasta de celulosa,así como los principales problemas que plantean algunos de sus constituyentes yalgunas soluciones biotecnológicas a estos problemas; ii) los objetivosperseguidos en la Tesis; iii) una descripción detallada de los materialesestudiados y los métodos analíticos empleados; iv) las referencias citadas en elprimer y tercer apartado; v) los resultados obtenidos y su discusión, que sepresentan en forma de publicaciones; vi) las principales conclusiones; y vii) unalista de tablas que se muestran como Anexos.3

1. IntroducciónINTRODUCCIÓN1.1. CULTIVOS LIGNOCELULÓSICOSLos cultivos lignocelulósicos incluyen especies tanto de origen agrícola comoforestal y poseen un gran interés industrial. Entre los principales usos de loscultivos lignocelulósicos se encuentra la producción de pasta de celulosa. Porotro lado, estos cultivos presentan un gran potencial como materia prima en elcontexto de las futuras biorrefinerías para la producción de biocombustibles yotros productos de interés, como alternativa al petróleo.La principal fuente de fibra de celulosa virgen utilizada actualmente en lafabricación de pasta de celulosa la constituyen los cultivos de fibras madereras,mientras que las fibras no madereras se utilizan en menor proporción. La ampliadisponibilidad y concentración de madera en zonas de fácil acceso, el elevadocontenido en fibras, el coste de manejo, transporte y facilidad dealmacenamiento, así como la estabilidad de la materia prima y sucomportamiento durante el proceso de obtención de celulosa, han apoyado eluso de la misma en la industria de la pasta de papel. Sin embargo, existe en laactualidad un renovado interés en el uso de plantas de origen no madererodebido a, entre otras razones, la gran disponibilidad de residuos agrícolas. Éstosconstituyen una fuente abundante de fibras de bajo coste, siendo a veces la únicafuente aprovechable de fibras en determinadas zonas geográficas,principalmente en países en vías de desarrollo. La gran variedad decaracterísticas, dimensiones fibrosas y composición química de estas fibras lesconfieren un gran potencial como materias primas (García Hortal 2007).Además, en los países desarrollados, se utilizan para la fabricación de pastas decelulosa para papeles especiales.1.1.1. Fibras procedentes de cultivos madererosLas fibras madereras provienen de especies vegetales que desarrollan untronco donde se acumulan preferentemente las mejores fibras. Las coníferasconstituyen el primer cultivo forestal a escala mundial para la obtención de pastade papel, aunque también existe un importante mercado de pastas de frondosas(Figura 1).Las coníferas presentan fibras largas (3 a 5 mm), que son óptimas para lafabricación de papeles de elevada resistencia mecánica. Las coníferas, entérminos económicos generales, son más valiosas que las frondosas, ya que sustroncos son más largos y rectos, su madera es uniforme, ligera y blanda, por loque es más fácil de trabajar, y presentan una mayor proporción de elementos5

1. Introducciónfibrosos que son más adecuados para la mayoría de las calidades papeleras(García Hortal 2007). Las principales coníferas usadas para la fabricación depasta de papel son la Picea y el pino.La madera de frondosas, por otro lado, es una madera más dura, de fibrascortas (entre 0,75 y 2 mm) que dan lugar a pastas menos uniformes. El papelfabricado con maderas de frondosas es más débil que los fabricados con maderasde coníferas pero su superficie es más lisa, y por lo tanto, es mejor para papel deescritura. Otra de las ventajas es que el crecimiento de las especies de frondosasutilizadas para la fabricación de pasta de papel es más rápido que el de lasconíferas, dando lugar a mayor cantidad de fibra en menos tiempo. Lasprincipales frondosas utilizadas en el sector papelero son el eucalipto, el chopo yel abedul.Figura 1. Ejemplos de especies madereras usadas para la producción de pasta de celulosa,incluyendo coníferas como la Picea (izquierda) y frondosas como el abedul (derecha).6

1. Introducción1.1.2. Fibras procedentes de cultivos agrícolasLas fibras procedentes de cultivos agrícolas constituyen una excelente materiaprima alternativa a las fibras madereras para la producción de pasta de celulosa.Uno de los hechos apremiantes que conducen a la utilización de materia primano maderera es su conocida abundancia que sobrepasa la utilización actual.En general, las fibras de plantas no madereras tienen una estructura menosdensa y más porosa, lo que implica un menor requerimiento de energía yproductos químicos para la separación de las fibras durante la producción depasta de papel. Además, presentan ciclos de crecimiento más cortos, alcanzandola madurez más rápidamente que las especies madereras y en muchos casos losrendimientos de pasta obtenidos son mayores (Tabla 1). Algunas pastas de fibraslargas no madereras, tienen propiedades superiores a las mejores pastas delmercado de coníferas, pues son extremadamente resistentes. El principalinconveniente de este tipo de materias primas es que la mayoría sólo estándisponibles en ciertas épocas del año.Tabla 1. Rendimiento promedio de algunas materias primas (Pierce 1991).EspeciesRendimientomateria seca (t/ha)RendimientoPasta (t/ha)Trigo 2,5 1,1Avena 1,6 0,7Centeno 2,2 1,1Arroz 3,0 1,2Caña de azúcar (bagazo) 9,0 4,2Bambú 4,0 1,6Miscanthus sinensis 12,0 5,7Canary grass 6,0 3,0Caña común 9,0 4,3Kenaf 15,0 6,5Cáñamo 12,0 6,7Frondosa de zona templada (abedul) 3,4 1,7Frondosa de crecimiento rápido (Eucalyptus) 15,0 7,4Conífera escandinava 1,5 0,7Conífera de crecimiento rápido 8,6 4,07

1. IntroducciónLas fibras no madereras se pueden clasificar en tres categorías: i) fibrasprocedentes del tallo de diversas plantas como lino, cáñamo, kenaf y yute, y dehojas como abacá y sisal; ii) residuos agrícolas como la paja de trigo, maíz yarroz o el bagazo de caña; y iii) hierbas silvestres como bambú o hierba elefante.Actualmente, las fibras no madereras representan una alternativa para laproducción de pasta de celulosa en países con baja disponibilidad de madera yen los que disponen de abundantes residuos agrícolas fibrosos o cultivos deplantas fibrosas no madereras. Así, el uso de estas fibras para la producción depasta de celulosa ha ido aumentando, especialmente en los países en vías dedesarrollo, como India, China y algunos países latinoamericanos.En los países desarrollados, las fibras no madereras se usan principalmentepara la producción de papeles especiales. En España, existen varias empresasque fabrican pasta de papel a partir de fibras no madereras. Entre ellas destaca laempresa CELESA que utiliza fibras liberianas (del tallo) de lino, cáñamo y yute,y fibras de hojas de sisal y abacá para fabricar pasta de celulosa para papelesespeciales de distintas características, tales como papeles para cigarrillos, filtrosespeciales o papeles dieléctricos (Figura 2). Dicha empresa ha suministrado lamayoría de las fibras y sus respectivas pastas de papel que se han estudiado enesta Tesis.Papel para bolsas de vacíoLinoYuteBolsas de téPapeles para filtrosCáñamoSisalPapelesdecorativosKenafAbacáPapeles para circuitoseléctricosPapel para cigarrillosFigura 2. Papeles especiales (izquierda) obtenidos de las pastas de papel fabricadas por laempresa CELESA (Tortosa, Tarragona) y sus principales materias primas (derecha).8

1. Introducción1.2. ESTRUCTURA Y COMPOSICIÓN QUÍMICA DE LOSMATERIALES LIGNOCELULÓSICOSLos materiales lignocelulósicos, incluyendo los productos de origen agrícolay forestal, representan la mayor fuente de energía y materia orgánica renovablesde la biosfera. Son materiales heterogéneos cuya estructura y composiciónquímica varían dentro de amplios límites y condicionan su utilización industrialy la posible aplicación de métodos biotecnológicos. Los principalescomponentes de estos materiales son los polímeros constituyentes de todas lasparedes celulares de materiales vegetales: celulosa, hemicelulosas y lignina(Figura 3), y una serie de compuestos de bajo peso molecular solubles en agua oen solventes orgánicos, así como pequeños contenidos en proteína y salesminerales (Fengel y Wegener 1984, Sjöström 1993).LigninaHemicelulosasCelulosaEnlaces dehidrógenoCelulosa Lignina HemicelulosasFigura 3. Representación esquemática de los principales constituyentes de la pared vegetalcorrespondiente a una angiosperma no leñosa (adaptado de Bidlack et al. 1992).9

1. Introducción1.2.1. CelulosaLa celulosa es el componente principal de las células vegetales, quecomprende aproximadamente del 10 al 20% del peso seco de las hojas, entre un43 y un 47% de la madera de coníferas, entre un 42 y un 44% de la madera defrondosas y el 90% del peso de las fibras de algodón (Streitwieser y Heathcock1983, Aitken et al. 1988). Estructuralmente, es un polímero lineal constituidopor unidades de -D-glucopiranosa unidas por enlaces glicosídicos (14), enlos que dos moléculas de glucosa se unen con eliminación de una molécula deagua entre dos hidroxilos de los carbonos 1 y 4. La configuración sólo esposible por la rotación de la unidad de glucosa siguiente del eje C 1 -C 4 del anillode piranosa, por eso la unidad de cadena de celulosa que se repite es la celobiosa(disacárido), con una longitud de 1,03 nm (Fengel y Wegener 1984, Sjöström1993, Sjöström y Westermark 1999). Los numerosos grupos hidroxilo favorecenla formación de enlaces de hidrógeno intra e inter-moleculares, formando cadaunidad de glucosa dos enlaces intramoleculares y uno intermolecular (Figura 4).Los enlaces de hidrógeno intermoleculares se establecen con otras cadenas queestán en el mismo plano, así como con cadenas en planos superiores e inferiores,de este modo, las cadenas de celulosa se unen dando lugar a la formación demicrofibrillas, y la unión de éstas entre sí a la fibra de celulosa, cuyos agregadosforman la pared celular (Lennholm y Henriksson 2007).Unidad celobiosaOOOHOHOHOHOHOHOOOHOOHOOH1OOOHOHOHO4OHOHOHOH1OOHOOHOOOOHOHOHOHO4HOHOOOHOOHOOH1OOOHOOHHOOHHOOH4OHOOHOOHO1OOOFigura 4. Estructura de la celulosa donde se muestra la unidad de celobiosa, la conformación (14) y los enlaces por puentes de hidrógeno intra e inter-moleculares.10

1. IntroducciónEn su estructura supramolecular, la celulosa se organiza en zonas cristalinas yzonas amorfas. Son los enlaces de hidrógeno inter-moleculares los que permitenuna estructura ordenada, esto es, una alta cristalinidad. En las zonas amorfas, elnúmero de enlaces por puentes de hidrógeno establecidos es menor y bastantemás desorganizado que en las zonas cristalinas, siendo por lo tanto la celulosaamorfa más fácil de disolver y más reactiva, pues la accesibilidad a los gruposhidroxilo es mayor (García Hortal 2007, Annergren 1996). Las propiedades delos materiales lignocelulósicos están relacionadas con el grado depolimerización de la molécula de celulosa, que es de al menos 15000 (Brett yWaldron 1996). La resistencia del papel es debida, en parte, a la resistenciaindividual de las cadenas de celulosa, que diminuye si estas se degradan.1.2.2. HemicelulosasLas hemicelulosas comprenden aproximadamente del 25 al 30% del peso secode la madera de coníferas, entre un 20 y 43% de la madera de frondosas, entreun 12 y 18% de las fibras liberianas de lino y un 12% de las fibras de hojas desisal (Aitken et al. 1988, Fengel y Wegener 1984, García Hortal 2007). Actúancomo matriz de soporte para las microfibrillas de celulosa y estructuralmenteson más complejas que la celulosa.Las hemicelulosas son polisacáridos heterogéneos constituidos por unacadena lineal de diferentes monosacáridos unidos principalmente por enlaces (14) y en algunos casos (13), de la que parten diversas ramificaciones(Sjöström 1993). Los principales monosacáridos que las constituyen incluyenpentosas (D-xilosa y L-arabinosa), hexosas (D-glucosa, D-galactosa, D-manosa,L-ramnosa y L-fucosa), y ácidos urónicos (ácido D-glucurónico y ácido D-galacturónico) (Figura 5) con un grado de polimerización entre 200 y 300,siendo más fáciles de disolver y de degradar que la celulosa (Sjöström yWestermark 1999). Contrariamente a la celulosa, la naturaleza de lashemicelulosas varía entre las diferencies especies (Tabla 2). En el caso de lasmaderas de coníferas se suele apreciar una mayor cantidad de hexosanos, comola manosa y galactosa, siendo predominantes los galactoglucomananos y losglucomananos (García Hortal 2007, Sjöström y Westermark 1999) aunquetambién se observan en las coníferas los arabinoglucuronoxilanos (Sjöström yAlén 1999). La xilosa es más abundante en las frondosas donde predominanpentosanos como los glucuronoxilanos (Fengel 1989, Sjöström 1993, Shimizu2001) aunque también se observan glucomananos (García Hortal 2007, Sjöströmy Alén 1999). En las plantas no madereras, las hemicelulosas presentan una granvariedad en su composición dependiendo de la especie, siendo en algunas, comoen el lino, predominantes la manosa y la galactosa (Morrison et al. 1999) y enotras, como en el kenaf, la xilosa (Han 1998, Neto et al. 1996).11

1. IntroducciónPENTOSAS HEXOSAS ÁCIDOS HEXURÓNICOSHHOHHHOHOHHHHOOHHOHHOHOHHHOOHHOHHOHOCOOHHHHOOHHOH-D-Xilosa-D-GlucosaÁcido -D-GlucurónicoOHHHHOHHHOOHOHHHHOHOOHHHOHOHHOHHH 3 COHOCOOHHHHOOHOHH-L-Arabinopiranosa-D-ManosaÁcido -D-4-O-MetilglucurónicoHOH 2 CHHOHOHOHHOHOH OHHHOHHHOOHOHHOHCOOHHHOHHHOOHOHH-L-Arabinofuranosa-D-GalactosaÁcido -D-GalacturónicoDESOXI-HEXOSASHOHHHOHHOOHOHHHOHHHOHHOOHOHH-L-Ramnosa-L-FucosaFigura 5. Monosacáridos componentes de las hemicelulosas (adaptado de Fengel y Wegener1984).12

1. IntroducciónTabla 2. Tipos y estructuras simplificadas de las principales hemicelulosas en diversosmateriales lignocelulósicos, X, xilosa; A, arabinosa; G, glucosa; Gal, galactosa; M, manosa;Ac, grupo acetilo; Gl, ácido 4-O-metilglucurónico (adaptado de García Hortal 2007)Tipo hemicelulosa Estructura simplificada PresenciaGlucuronoxilanosX- X - X - XAcGl7Frondosas, plantas no madererasGlucomananosG -M -G -M -MConíferas, frondosasGalactoglucomananosG-M -M -MGal AcConíferasArabinoglucuronoxilanosX -X -X- X -X 5GlA2Coníferas, plantas no madererasLas hemicelulosas, con estructura ramificada y amorfa, son muy hidrofílicas ydesempeñan un papel fundamental en el proceso de fabricación de papel alpromover el hinchamiento de la fibra y aumentar su plasticidad, flexibilidad ycapacidad de enlace, con la consiguiente mejora de la densidad de la hoja. Sinembargo, durante el secado de la pasta también tienden a mantener dura o rígidala fibra, lo que puede impedir la subsiguiente rehidratación de la pasta (GarcíaHortal 2007).1.2.3. LigninaDespués de la celulosa, la lignina es el polímero más abundante en el mundovegetal, representando entre un 25 y un 33% de la madera de coníferas y entreun 18 y un 34% de la madera de frondosas (Aitken et al. 1988). En el caso de lasplantas no madereras hay un menor porcentaje de lignina con respecto a lasespecies madereras, con un 8-9% para fibras de hojas (abacá y sisal), entre un 3-13% para fibras liberianas (lino, cáñamo, 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 cañas13

1. Introducción(azúcar, bambúes) (García Hortal 2007). La lignina actúa como aglomerante delas fibras debido a su carácter hidrófobo siendo una de las moléculas orgánicasmás recalcitrantes.Estructuralmente, la lignina es un heteropolímero aromático con unaestructura tridimensional irregular, constituida por unidades de fenilpropano condiferentes patrones de substitución y unidas por diferentes tipos de enlaces, quevarían considerablemente entre las especies vegetales e incluso dependiendo dela edad (Freudenberg y Lehmann 1963), de la parte del árbol/planta (Bland1966), tipo de células (Fergus y Goring 1970, Hardell et al. 1980a, 1980b) y dellugar de la pared celular donde se sintetice (Fergus y Goring 1970, Fukushima yTerashima 1991, Christierini et al. 2005), por lo que la lignina no puede serdescrita por una fórmula simple.Los precursores de la lignina son los alcoholes p-hidroxicinamílicos (Figura6), que incluyen los alcoholes p-cumarílico (4-hidroxicinamílico, I), coniferílico(4-hidroxi-3-metoxicinamílico, II) y sinapílico (4-hidroxi-3,5-dimetoxicinamílico, III), que difieren entre sí en el número de grupos metoxilosustituyentes. Estos precursores se sintetizan a su vez a partir de la fenilalanina através de la ruta de los ácidos cinámicos (Higuchi 1997, Boerjan et al. 2003,Freudenberg y Neish 1968, Adler 1977, Ralph et al. 2004). Recientemente, se hadescrito la existencia de otros precursores de la lignina, tales como derivadosacilados (acetatos y/o p-cumaratos) de los correspondientes alcoholes p-hidroxicinamílicos (IV y V) (del Río et al. 2004, 2007, 2008a, 2008b, Martínezet al. 2008) observados en diversas plantas angiospermas, así como alcoholesdihidroxicinamílicos (VI), o aldehídos cinamílicos (VII), observados en lalignina de especies modificadas genéticamente (Ralph et al. 1997, 1998,Sederoff et al. 1999). Su deshidrogenación oxidativa, catalizada por peroxidasaso lacasas en presencia de peróxido de hidrógeno u oxígeno, respectivamente,conlleva a la formación de radicales fenoxilo estabilizados por resonancia queluego se acoplan entre sí y con el polímero creciente de lignina mediantediversos tipos de enlaces (Figura 7).Aunque la variedad de uniones para formar el polímero de lignina es amplia(Figura 8), se pueden diferenciar dos tipos: uniones de tipo éter y uniones detipo carbono-carbono. La formación de enlaces éter-alquil-arílico es la másfavorable termodinámicamente, como es el caso del enlace -O-4, en el que seencuentran involucrados la posición del monolignol radical y el radicalfenoxilo del polímero de lignina creciente. En menor proporción existen unionesde tipo aril-aril éter, como por ejemplo la unión 4-O-5. Los enlaces de tipocarbono-carbono, conocidos también como enlaces condensados, son másdifíciles de romper que los de tipo éter, e incluyen las uniones de dos cadenasalifáticas (- resinol), la unión de un carbono de un anillo bencénico con el deuna cadena alifática (-1 y -5 fenilcumarano) y las uniones entre carbonos de14

1. Introduccióndos anillos bencénicos (5-5). Se ha descrito que el enlace 5-5 no se encuentratal cual, sino en forma de trímero, ya que incorpora una nueva unidad medianteun enlace -O-4 y un enlace -O-4, dando lugar a una estructura de tipodibenzodioxocina (Karkunen et al. 1995). Igualmente, estudios recientes indicanque la mayoría de las uniones -1 se encuentran en forma de espirodienonas(Zhang y Gellersted 2001, Zhang et al. 2006).OHOHOHOOOMe(MeO)OMe(MeO)(OMe)OHOHOHOHI II III IVOHOOHHOO(MeO)(OMe)HOOMe(MeO)(OMe)OHOHOHV VI VIIFigura 6. Estructuras de precursores de la lignina: I, alcohol p-cumarílico; II, alcoholconiferílico; III, alcohol sinapílico; IV, derivado acetilado de los alcoholes p-hidroxicinamílicos; V, derivados p-cumaroilados de los alcoholes p-hidroxicinamílicos; VI,alcohol 5-hidroxiconiferílico y VII, aldehídos correspondientes a los alcoholes p-hidroxicinamílicos.15

1. IntroducciónOHPeroxidasaLacasaOHOHOHOHOH-(e - + H + )OHOMeOOMeOOMeaOOMeOOMeOOMe(MeO)Lignina(MeO)LigninaHOHOOMeOxidaciónHOOOMeAcoplamientoradicalar(MeO)HOOLignina(MeO)HOROOMeROHOMeLignina(MeO)OHOMe (MeO)OMe (MeO) OMeOOb(MeO)OHOMeFigura 7. Síntesis de la lignina: (a) deshidrogenación del alcohol coniferílico y formasresonantes del radical fenoxilo (adaptado de Adler 1977) y (b) mecanismo de la unión de losmonolignoles libres al polímero de lignina (Freudenberg y Neish 1968).La cantidad de lignina, su distribución a través de las paredes celulares y laestructura básica de la misma, difieren según su origen entre coníferas,frondosas y fibras no madereras. En la lignina de las coníferas la estructura quese repite predominantemente es la unidad guayacilo (G), que contiene un únicogrupo metoxilo en el anillo de fenilpropano y deriva del alcohol coniferílico(más de 95% de las unidades estructurales). En el caso de la lignina de lasmaderas de frondosas, hay predominantemente dos unidades que se repiten, launidad guayacilo (G) y la unidad siringilo (S), conteniendo esta última dosgrupos metoxilo por núcleo de fenilpropano y deriva del alcohol sinapílico(Parhan 1983, Sarkanen y Hergert 1971, Lin y Dence 1992). Por otro lado, lalignina de fibras no madereras presenta unidades del tipo p-hidroxifenilo (H),procedentes del alcohol p-cumarílico, y unidades S y G, en proporcionesvariables dependiendo de la planta. Las unidades G, al contrario que las S,tienen un único grupo metoxilo y la posición C-5 está libre y disponible para laformación de enlaces carbono-carbono, por lo que ligninas con mayor cantidadde unidades G tienen una estructura más condensada y por lo tanto la lignina sedegrada con mayor dificultad. En la Figura 9 se muestran modelos estructuralesdel polímero de lignina de coníferas y frondosas, y en la Figura 10 se muestranmodelos estructurales del polímero de lignina de algunas plantas no madereras.16

1. IntroducciónHOHOMeO O5’4’6’3’1’2’HO6’1’MeO 5’2’OH4’ 3’ OOMe612OMe612OMe21’1 36’ 2’MeO543OMeOMe543OMe654O5’4’3’OMeOOOMeO-O-4-O-4 4-O-5 OMeHOMeO651’6’5’4’ O1234O2’3’OMeOMeMeOHO65HO14O 231’6’2’5’3’4’OMeOMeOOMeMeOO546123OMeOO’’’2’1’3’6’4’5’OOMe-5 /-O-4 fenilcumarano-1 - /-O- /-O- resinol2166’1’2’MeOMeO36’5’’4’’54O1’’2’’3’’5’4’O3’OHOMeMeOOOMe34251 MeO6 O ´HO1´6´ 2´6''5''1''2''3''4''O´´OHOMeOOMeMeO5´4´O3´OMe5-5 / -O-4 / -O-4 dibenzodioxocina-1 /-O- espirodienonaFigura 8. Enlaces tipo éter y carbono-carbono presentes en el polímero de lignina.17

1. IntroducciónAB(a)Figura 9. Modelos del polímero de lignina en maderas: (a) conífera (Picea) (Brunow 2001) y(b) frondosa (álamo) (Boerjan et al. 2003).(b)18

1. IntroducciónOMeOHHOOMeOOOMe OOHOMe OHOHOOMeOOO MeO OMeOOMeO OMeOOHHOOMeOO OHOMeO OMeOO OHOOMeOOMe OHOHMeO OMeHOOO OMeOOMeOHMeOHOOHO OHOO MeO OMeOHOO OHOOMe OOOHOMeMeO OMeO OOOHOOMe OOOHOMeO MeO OMeOOHOHOOMeOHOOMe OHOOMeO OMeHOO OMeOOOMeOHMeOO OMeOHO OHOOMeMeO OO OHOMeOHOOOMeOOOHOOMeOHOMeOMeOOOOMeOOHOOMeOOHOOMeOOMeHOOMeOOOHOMeOHOOMeO OHOMeOHMeOOOOMeOOHOOHO OO OOMeOMeO OOHMeOMeO HOO OMeOOHOHOHMeOMeOHOOMeHOOMe(a)(b)Figura 10. Modelos del polímero de lignina en plantas no madereras: (a) kenaf y (b) abacá(del Río et al. 2008a).19

1. IntroducciónLa lignina aparece también asociada a los polisacáridos en la pared celular yes esta asociación la que determina la rigidez y la resistencia estructural delmaterial. Las hemicelulosas están asociadas a la lignina principalmente a travésde las unidades de arabinosa, xilosa y galactosa por enlaces de tipo glicosídico,éter bencílico y éster bencílico formando complejos lignina-polisacáridos(Watanabe 2003).1.2.4. Componentes de bajo peso molecularAdemás de los carbohidratos (celulosa y hemicelulosas) y lignina, existen enlos materiales lignocelulósicos pequeñas cantidades de componentes que noinfluyen en la estructura morfológica de las células pero que tienen graninfluencia en el procesamiento de estos materiales. Algunos de estoscomponentes protegen a la madera de los insectos y son responsables de sucolor, olor y gusto. Atendiendo a su solubilidad se pueden dividir en compuestosextraíbles en solventes apolares, que incluyen los extraíbles lipofílicos, ycompuestos extraíbles en disolventes polares (extraíbles hidrofílicos), así comodiversos compuestos insolubles tales como sustancias proteicas, pécticas y denaturaleza inorgánica (García Hortal 2007, Hillis 1962, Fengel y Wegener 1984,Rowe 1989, Sjöström 1993).Los extraíbles lipofílicos (Figura 11) incluyen típicamente alcanos (a),alcoholes grasos (b), aldehídos (c), ácidos grasos (d), esteroles (e), ácidosresínicos (f), ceras (ésteres de ácidos grasos con alcoholes de cadena larga, g) yglicéridos (ésteres de ácidos grasos con glicerol, h). Los esteroles puedenencontrarse libres o esterificados con ácidos grasos (ésteres de esteroles, i) ytambién pueden estar formando glicósidos y acilglicósidos (Gutiérrez y del Río2001), siendo el más abundante el 3-D-glucopiranósido (j).Los extraíbles lipofílicos afectan negativamente al proceso de fabricación depasta de celulosa así como al producto final, formando depósitos insolublescomúnmente denominados depósitos de pitch, que se describen más adelante enel apartado 1.4. Debido a su alto grado de pegajosidad, los esteroles libres yconjugados se encuentran en el origen de muchos depósitos de pitch (Back yAllen 2000, del Río et al. 1998, 2000). El estudio de los compuestos extraíbleslipofílicos de cada una de las materias primas constituye un requisitofundamental para identificar los compuestos que originan los depósitos de pitchy diseñar estrategias adecuadas para su control.20

1. IntroducciónabOHOOcHdOHHOeCOOHfFOOgCO-O-CH 2CO-O-CHCO-O-CH 2hOCH 2 OHOiOHOOHOHOjFigura 11. Estructuras de compuestos representativos de las principales familias deextraíbles lipofílicos: (a) pentacosano, (b) docosanol, (c) octacosanal, (d) ácido palmítico, (e)sitosterol, (f) ácido abiético, (g) octacosanil hexadecanoato, (h) trilinoleína, (i) sitosterillinoleato, (j) sitosteril 3-D-glucopiranósido.21

1. IntroducciónPor otro lado, los extraíbles polares engloban diferentes compuestos fenólicoslibres de bajo peso molecular (Figura 12), los cuales incluyen precursores de lalignina (ácidos p-hidroxicinámicos y aldehídos p-hidroxicinamílicos), ácidosbencenocarboxílicos relacionados (ácido p-hidroxibenzoico, vainíllico ysiríngico), aldehídos y cetonas aromáticas (p-hidroxibenzaldehído, vainillina,siringaldehído y propioguayacona), e incluyen taninos hidrolizables (ésteres delácido gálico y sus dímeros), flavonoides (estructuras derivadas del anillo deflavona, 2-fenilbenzopirona) y taninos no hidrolizables (varias unidades deflavonoides condensadas). Además de incrementar el consumo de reactivosdurante la cocción, estos compuestos pueden dificultar las reacciones depasteado impidiendo la difusión de los reactivos en la materia prima, y lostaninos, cuando están presentes en cantidades importantes, forman complejoscoloreados con cationes metálicos afectando el color de las pastas de papel y sublanqueabilidad (García Hortal 2007).HOOHOOOHOOOOOOOHOHOHOHa b c dHOOOHOOHOHeOfFigura 12. Estructuras de compuestos representativos de los compuestos extraíbles polares:(a) Ácido siríngico, (b) ácido p-hidroxibenzoico, (c) vainillina, (d) acetosiringona, (e) ácidogálico y (f) 2-fenilbenzopirona.22

1. Introducción1.3. UTILIZACIÓN DE CULTIVOS LIGNOCELULÓSICOS:PRODUCCIÓN DE PASTA DE CELULOSALa fabricación de pasta de celulosa consiste básicamente en la separación delas fibras de celulosa de la madera u otros materiales fibrosos a través deprocesos mecánicos y/o químicos (Fengel y Wegener 1984, Sjöström 1993). Secree que la fabricación de papel tuvo su origen en China hacia el año 100 d.C. ypara su fabricación se utilizaban trapos, cáñamo, paja y hierba como materiasprimas, que se golpeaban contra morteros de piedra para separar la fibraoriginal. Aunque con el tiempo ganó terreno la mecanización, hasta el siglo XIXsiguieron utilizándose los métodos de producción por lotes y las fuentes de fibraagrícolas. Las primeras máquinas continuas de papel se patentaron a finales delsiglo XIX y principios del siglo XX. Entre 1844 y 1884 se desarrollaron losprimeros métodos para la obtención de pasta a partir de madera, una fuente defibra más abundante que los trapos o hierbas; estos métodos implicaban laabrasión mecánica y la aplicación de diversos procedimientos químicos. Lafabricación de papel fue una labor artesana e individualizada, pero con losdescubrimientos de la ciencia y los avances tecnológicos, así como con eldesarrollo y expansión de la cultura, la industria de fabricación del papel sedesarrolló a un ritmo acelerado. La industria de pasta celulósica muestra aún unatendencia creciente en su producción, según los datos de la FAO (FAO 2004) dela Tabla 3.Tabla 3. Estadísticas sobre la capacidad de producción de papel en los principales paísesproductores (2003-2008) (FAO 2004)2003 2004 2005 2006 2007 2008Capacidad total , 1000MtPaíses desarrollados 244169 247669 251387 254304 255786 256589Norteamérica 114550 114325 115062 115405 115405 115405Europa 91894 95866 98824 101360 102827 103620Oceanía 4049 4143 4208 4246 4261 4271Otros 33676 33335 33293 33293 33293 33293Países en desarrollo 21201 21774 22434 22964 23296 23493África 2681 2688 2721 2865 2865 2872América Latina 13181 13469 13841 14052 14219 14261Asia 5339 5617 5872 6047 6212 636023

1. IntroducciónEl proceso de producción de pasta de celulosa comprende fundamentalmenteel proceso de pasteado y el proceso de blanqueo. El proceso de pasteado tienepor objeto separar las fibras de celulosa del resto de los componentes de lamadera, fundamentalmente de la lignina ya que las fibras de celulosa seencuentran cementadas por ella. Por otro lado, el blanqueo de la pasta tiene porobjeto disolver o modificar la lignina residual que no se elimina durante elpasteado, para mejorar las propiedades de la pasta y consecuentemente delproducto final.1.3.1. Procesos de pasteadoDependiendo de las características de las fibras, el tratamiento aplicado paradestruir o debilitar los enlaces interfibras varía, con la finalidad de obtener unapasta de características adecuadas y el mayor rendimiento posible. Los procesosde obtención de pasta de papel se clasifican básicamente en mecánicos yquímicos. Combinaciones de éstos dan lugar a procedimientos intermedios osemiquímicos.Procesos mecánicosEl pasteado mecánico tiene como objeto la separación física de las fibras,realizándose el desfibrado por fragmentación mecánica, utilizando molinos yrefinadores de discos. La fabricación de pastas mecánicas ofrece la ventaja dedar como resultado rendimientos elevados (hasta un 98% del material inicial),obteniéndose pastas ventajosas para algunos tipos de papel por su rigidez,volumen y opacidad (García Hortal 2007). Sin embargo, como en este proceso lalignina sólo se ablanda (no se disuelve), el alto contenido en lignina va endetrimento de la calidad del papel ya que las fibras muy lignificadas son rígidas,poco flexibles, no están bien unidas entre sí, proporcionando papeles con bajascaracterísticas de resistencia y muy sensibles al envejecimiento óptico.Procesos químicosEn el pasteado o cocción química, la deslignificación se lleva a cabo con laayuda de agentes químicos ácidos o básicos, en digestores o reactores a altastemperaturas y presiones. La pasta se produce con disolución de la lignina quese encuentra entre las fibras del material lignocelulósico y los productos dedegradación se disuelven en la lejía de la cocción. En el pasteado químico, seeliminan muchos de los componentes no fibrosos de la materia prima y losrendimientos son normalmente del 35 al 65%, sin embargo, la pasta se blanqueamejor y el producto es más resistente y de mejor calidad que en el caso de losprocesos mecánicos (Sjöström 1993).24

1. IntroducciónLos procesos de pasteado químico pueden realizarse en condiciones alcalinas,como el pasteado a la sosa y el proceso kraft, o en condiciones ácidas como elpasteado al sulfito. Otro tipo de procesos utiliza solventes orgánicos (pasteadoorganosolv) (Gilarranz et al. 1999).El proceso a la sosa es el más antiguo y el más simple de los procesosquímicos alcalinos. En este proceso, la fibra se somete a un proceso de coccióncon sosa cáustica y vapor a alta presión y temperatura. El hidróxido de sodio esun producto muy útil para la deslignificación de materias primas vegetales,principalmente de maderas, pajas de cereales y plantas fibrosas en general. Eneste proceso, se puede utilizar antraquinona (AQ) como catalizador ya quepresenta dos efectos fundamentales como son la aceleración del proceso dedeslignificación alcalino y la estabilización de los carbohidratos, mejorando losrendimientos respecto al proceso convencional en las mismas condiciones deoperación (Abarca y Blanco 2008).El proceso kraft para la obtención de pasta de papel es un proceso químicoalcalino que deriva del proceso a la sosa. En este proceso, además de hidróxidode sodio se utiliza sulfuro sódico, siendo estos agentes de cocción conocidoscomo lejías blancas. El proceso se lleva a cabo en digestores que pueden sertanto discontinuos como continuos, en los que se introducen las astillas junto alas lejías blancas llevándose a cabo la cocción a elevada temperatura (150-170ºC) y presión. Generalmente el proceso tiene lugar con una concentración dereactivos del 16-20% (expresados como peso de Na 2 O, en relación al peso de lamadera). Este tipo de pasteado permite obtener pastas con una gran resistencia,aunque con menor rendimiento (entre un 40 y 60%), ya que se elimina muchacantidad de lignina (hasta el 90%) (García Hortal y Colom 1992, Santos et al.1997). La ventaja de este proceso es que requiere tiempos de cocciónrelativamente cortos pues el sulfuro acelera la deslignificación reduciendo ladegradación del material celulósico y produciendo así pastas de mejor calidad.Para este proceso, se pueden utilizar todo tipo de maderas, aunque los mejoresresultados se obtienen con maderas de frondosas.El proceso al sulfito es un proceso químico ácido donde se utilizan sulfitos ybisulfitos para la deslignificación. Es un proceso más fuerte que el procesoalcalino y permite una mejor separación de la celulosa. Este proceso estálimitado en cuanto al tipo de materia prima, pues no se pueden utilizar maderasde coníferas ya que a pH bajos los fenoles y los ácidos resínicos se condensancon la lignina formando complejos insolubles y coloreados que manchan lapasta. El licor de cocción es una disolución de ácido sulfuroso (H 2 SO 3 ) ybisulfito de calcio (Ca(HSO 3 ) 2 ), que se prepara disolviendo dióxido de azufre enagua y haciéndola reaccionar con CaCO 3 . Los digestores operan a temperaturascomprendidas entre los 125 y 180ºC según la aplicación que se quiera dar alproducto final (papel, cartón, etc.), obteniéndose rendimientos entre el 40 y25

1. Introducción60%. En estos procesos también se degradan los hidratos de carbono por roturade los enlaces glicosídicos, lo que provoca una disminución del grado depolimerización todavía mayor que en los procesos kraft siendo la pastaresultante menos resistente, pero por lo contrario estas pastas son más fáciles deblanquear. El método al sulfito ha sido relegado en parte por el proceso kraft(Bryce 1990).Alternativamente, se han desarrollado los procesos organosolv que utilizansolventes orgánicos para la deslignificación. Estos procesos presentan unamayor selectividad y por lo tanto, dan lugar a rendimientos mayores. Por otrolado, permiten la utilización de cualquier materia prima fibrosa (coníferas,frondosas y plantas no madereras) dando lugar a la obtención de pastas con bajocontenido en lignina que pueden ser blanqueadas sin el uso de compuestosclorados. Se han empleado multitud de disolventes orgánicos (etanol, metanol,butanol, alcohol bencílico, glicerol, glicol, etilenglicol, trietilenglicol, fenol,acetona, ácido fórmico, ácido acético, dioxano, dimetilsulfóxido,hexametilendiamina, etc.) puros o en disolución acuosa, con la adición o no decatalizadores. Los elevados precios de los reactivos, la dificultad en surecuperación y en muchos casos su elevada toxicidad, ha favorecido el uso dealcoholes alifáticos de bajo peso molecular (etanol y metanol) como solventespara los procesos organosolv (Herrero et al. 2002). Estos solventes combinan sualta velocidad de deslignificación en condiciones de operación favorables y sufácil recuperación. Sin embargo, en general, las propiedades de resistencia de laspastas organosolv son inferiores a las pastas kraft.1.3.2. Procesos de blanqueoEn el proceso de blanqueo se trata químicamente la pasta de celulosa paraeliminar o modificar la lignina residual que queda después del proceso decocción. Los componentes coloreados de la lignina se degradan, disuelven o sedecoloran (Sjöström 1993). El proceso de blanqueo se lleva a cabo hasta elpunto de blancura que se pretende, por lo que el número de etapas dependerá dela calidad de la pasta que se desee obtener (Figura 13). Los reactivoscomerciales más utilizados para el blanqueo son el cloro gas, el hipoclorito, elperóxido de hidrógeno y el dióxido de cloro; y el álcali utilizado es el hidróxidode sodio que se usa en la operación de extracción alcalina.26

1. IntroducciónFigura 13. Diferentes grados de blancura de una pasta de celulosa.El blanqueo ha sido la etapa de la producción de pasta de celulosa que hasufrido más cambios durante los últimos años. La decisión de eliminar el cloromolecular y, en algunos casos también el dióxido de cloro, de las secuencias deblanqueo se debe a la necesidad de reducir las emisiones de compuestosclorados orgánicos, de haluros orgánicos absorbibles y dioxinas en los efluentesde las plantas de blanqueo. El desarrollo de leyes más restrictivas con respecto alos procesos contaminantes ha llevado a una parte importante de la industriaeuropea de pasta y papel a introducir secuencias de blanqueo totalmente libresde cloro (blanqueo TCF, totally chlorine free) (Brooks et al. 1994). Estassecuencias incluyen blanqueo con peróxido de hidrógeno, oxígeno y ozono. Otraparte de la industria papelera mundial, ha eliminado el cloro elemental perocontinúa utilizando dióxido de cloro en el blanqueo (blanqueo ECF, elementalchlorine free).El desarrollo del blanqueo con oxígeno ha sido bastante lento por ladegradación de la celulosa y demás polisacáridos de la madera. Las ventajas delperóxido de hidrógeno se apoyan en su facilidad de manipulación y aplicación,su versatilidad y la naturaleza relativamente inocua de los productos de reacción.La novedad de las secuencias de blanqueo TCF obliga a solucionar nuevosproblemas que surgen al introducir métodos de blanqueo menos agresivos y quese describen en el apartado siguiente.En España, diversas empresas productoras de pasta de papel, incluyendoENCE y CELESA (que han suministrado pastas de papel para la presente Tesis),han realizado un considerable esfuerzo de inversión y modernización de susfábricas con objeto de adaptarlas a las modernas tecnologías ECF y TCF.27

1. Introducción1.4. PROBLEMÁTICA PLANTEADA POR LA PRESENCIA DELIGNINA Y LÍPIDOS EN LA PRODUCCIÓN DE PASTA DECELULOSAUna parte de los problemas originados durante la producción de pasta decelulosa está relacionada con los compuestos extraíbles lipofílicos de losmateriales lignocelulósicos. Estos compuestos causan tanto problemasmedioambientales como problemas técnicos durante el proceso de producción.Entre los compuestos lipofílicos más problemáticos están los ácidos grasoslibres, ácidos resínicos, ceras, alcoholes, esteroles tanto libres comoesterificados, glicéridos, cetonas y otros compuestos (Hillis 1962, Fengel yWegener 1984, Rowe 1989, Gutiérrez et al. 1999). Durante el proceso deproducción de pasta de celulosa los compuestos lipofílicos se liberan formandopartículas coloidales que pueden unirse y formar gotas que luego se depositan enla pasta o en la maquinaria formando los llamados “depósitos de pitch” (Figura14). La formación de estos depósitos da lugar a importantes pérdidaseconómicas como consecuencia de pastas contaminadas, paradas en laproducción, así como por el coste de los aditivos químicos utilizados para elcontrol del pitch (Hillis 1989, Allen 2000). Además, algunos compuestoslipofílicos de los materiales lignocelulósicos también tienen un impacto negativosobre el medio ambiente, considerándose algunos de ellos como primera fuentede toxicidad cuando se liberan en los vertidos (Ali y Sreekrishnan 2001, Rigol etal. 2004). Esto es especialmente importante en los procesos modernos donde elblanqueo con cloro ha sido sustituido por el blanqueo libre de cloro elemental(ECF) o totalmente libre de cloro (TCF). El blanqueo ECF evita los problemasasociados a la formación de compuestos clorados producidos en el blanqueo concloro. Sin embargo, algunos de los extraíbles lipofílicos que son destruidos porel dióxido de cloro no se eliminan en el blanqueo TCF, ya que estas secuenciasque utilizan oxígeno y peróxido de hidrógeno no afectan prácticamente a lafracción lipídica de las materias primas.La problemática del pitch es muy compleja porque varía con la materia primaasí como con el proceso empleado para la fabricación de pasta y papel. En elcaso de las pastas mecánicas, los depósitos de pitch muestran una composiciónsimilar a los extractos lipofílicos de la materia prima. En el caso de las pastasalcalinas, sólo algunos de los compuestos extraíbles presentes en la materiaprima sobreviven al proceso de cocción. En condiciones alcalinas los ésteres deglicerol se saponifican y los ácidos grasos y resínicos se disuelven. Los ésteresde esteroles, los esteroles libres y las ceras, se saponifican más lentamente quelos ésteres de glicerol, no forman jabones solubles como en el caso de los ácidoslibres, por lo que tienen tendencia a depositarse (Gutiérrez et al. 2001).28

1. IntroducciónFigura 14. Imagen de una gota de resina en el árbol (izquierda) y de un depósito de pitchen una pasta kraft TCF (cedidas por María Jesús Ortega y Javier Romero, respectivamente).Por otro lado, la lignina está también relacionada con la problemáticaexistente en la producción de pasta de celulosa ya que la variabilidad en sucomposición y estructura influye decisivamente en el proceso dedeslignificación. Por otro lado, la formación de compuestos oxidados de lalignina durante el proceso de pasteado (lignina residual) es responsable del coloroscuro de las pastas. La fabricación de pastas de papel mediante tecnologíasmenos contaminantes ha traído consigo nuevos problemas en el blanqueo de lapasta, que no se daban al utilizar reactivos más agresivos (aunque también máscontaminantes) y/o en sistemas con un menor grado de cierre en los circuitos.De momento ni el oxígeno ni la combinación de oxígeno y peróxido puedenigualar la eficacia de la cloración para la eliminación de los productos derivadosde la lignina, responsable del color de las pastas.1.5. BIOTECNOLOGÍA EN EL SECTOR DE LA PASTA DE CELULOSALa producción de pasta y papel ha sido tradicionalmente un proceso industrialcon un fuerte impacto medioambiental. El gran incremento en la demanda depapel ha agravado el impacto negativo sobre el medio ambiente, por lo que sehan desarrollado leyes más restrictivas con respecto a los procesoscontaminantes. Por consiguiente, las empresas papeleras han tenido que realizarun considerable esfuerzo de inversión y modernización de sus fábricas conobjeto de adaptarlas a tecnologías más limpias y además, con un mayor grado decierre en los circuitos para reducir los efluentes líquidos. La biotecnologíaaplicada a este sector ofrece nuevas posibilidades de utilizar métodos biológicos29

1. Introducciónbasados en el uso de hongos y enzimas para reducir o remediar el impactomedioambiental, reduciendo el consumo de reactivos químicos, así como elgasto energético durante la fabricación de pasta de papel.Durante los últimos años, el número de aplicaciones enzimáticas en laindustria de la pasta de celulosa ha aumentado considerablemente, y varias hanalcanzado o se están acercando a su uso comercial. Éstas incluyen el uso dexilanasas para ayudar al blanqueo, la deslignificación directa con enzimasoxidativas, el ahorro de energía de refino con celulasas, así como la reducciónde depósitos de pitch con lipasas (Bajpai 1999, 2006). Además de las enzimas,los tratamientos microbianos también tienen una potencial aplicación paraaumentar la eficiencia en la fabricación de pasta de celulosa, para la reducciónde los problemas de pitch y la mejora en la reutilización de las aguas delproceso.En la presente Tesis, se incluye el estudio de enzimas para dos de estasaplicaciones, como son el blanqueo de la pasta de papel y el control del pitch,que se mencionan a continuación.1.5.1. Degradación enzimática de la ligninaEl uso de enzimas en la industria papelera ha crecido rápidamente a partir demediados de los años 80. Las enzimas más utilizadas en el blanqueo de laspastas son enzimas hidrolíticas como las xilanasas, que se utilizan para limitar eluso de cloro en los procesos de blanqueo de la pasta (Viikari et al. 1994). Lasxilanasas no actúan directamente sobre la lignina, sino catalizando la hidrólisisde los xilanos que se encuentran entre las microfibrillas de la celulosa y lalignina. Sin embargo, enzimas de tipo oxidoreductasa (lacasas y peroxidasas)tienen mayor potencial que las xilanasas porque actúan directamente sobre lalignina. Durante años se concedió mayor atención a las peroxidasasligninolíticas que a las lacasas en la degradación de la lignina y en el desarrollode aplicaciones biotecnológicas (Paice et al. 1995) ya que los bajos potencialesredox de las lacasas (0.3 a 0.8 V) comparados con los de las peroxidasasligninolíticas (>1 V) sólo permiten a las lacasas la degradación directa decompuestos fenólicos de bajo potencial redox, que constituyen únicamente un20% del total de la lignina (Kawai et al. 1987a, 1987b). El interés por las lacasascomo biocatalizadores industriales en la producción de pasta de papel se haincrementado enormemente tras el descubrimiento de compuestos mediadoresque amplían la acción de la lacasa a sustratos no fenólicos, lo que aumenta elpotencial en la degradación de la lignina y de otros compuestos aromáticos (Cally Mücke 1997). En la Figura 15 se pueden observar las estructuras de estas tresenzimas.30

1. Introducción(a)(b)(c)Figura 15. Enzimas de interés en la industria de la pasta y papel: (a) xilanasa, (b) lacasa y (c)peroxidasa versátil.Las peroxidasas catalizan la oxidación de una gran variedad de compuestostanto orgánicos como inorgánicos en presencia de peróxidos. Hay dos tipos deperoxidasas ligninolíticas: la lignina peroxidasa (LiP) y las manganesoperoxidasas (MnP) (Tien y Kirk 1983, Glenn et al. 1983, Kuwahara et al. 1984).La LiP oxida compuestos aromáticos de alto potencial redox, como el alcoholveratrílico (alcohol 3,4-dimetoxibenzílico) y dímeros modelo de lignina de tipono fenólico. Esta enzima es una glicoproteína con hierro protoporfirínico IXcomo grupo prostético, dependiente de H 2 O 2 para su actividad. Inicialmente esoxidada por peróxido de hidrógeno, oxidando núcleos aromáticos de la moléculade lignina (fenólicos y no fenólicos), generando radicales catiónicos. Estosinteractúan espontáneamente con nucleófilos (principalmente H 2 O) y conoxígeno molecular, generando una “combustión enzimática” donde los enlacesC-C e C-O se rompen, despolimerizando la lignina y abriendo los anillosaromáticos. Las MnP son glicoproteínas con hierro protoporfirínico IX comogrupo prostético y dependiente de H 2 O 2 para su actividad, pero la oxidación poresta enzima es también dependiente de la disponibilidad de iones manganeso.Un tercer tipo de peroxidasa ligninolítica, la VP (peroxidasa versátil), se hadescrito por primera vez en Pleurotus eryngii (Martínez et al. 1996, Ruiz-Dueñas et al. 1999a, Camarero et al. 1999) y se caracteriza por combinarpropiedades catalíticas de las otras dos peroxidasas ligninolíticas (Ruiz-Dueñaset al. 1999b) pudiendo oxidar lignina y compuestos de manganeso. En la Figura16 se puede observar una vista axial de la región del hemo de la VP donde semuestra los tres sitios de la oxidación y el ciclo catalítico de ésta enzima. La VPse ha utilizado en esta Tesis en ensayos de deslignificación conpolioxometalatos (POMs).31

1. IntroducciónAlcoholveratrílico(VA)Trp164·Hélice BaguaGlu40Glu36Mn 2+ROOHROHVP[Fe 3+ ]ABTS·+VA·Mn 3+VAMn 2+C-IIB[Fe 3+ Trp·]Grupo HemoAsp175ABTSC-IA[Fe 4+ =O P·]ABTS ABTS·+ VAMn 2+ Mn 3+ C-IIA[Fe 4+ =O]VA·ABTS(fenoles)C-IB[Fe 4+ =O Trp·](a)Figura 16. Vista axial de la región del hemo de la VP que muestra los tres sitios de laoxidación: Mn 2+ (que incluye tres residuos acídicos de amino-ácidos), ABTS (en el límite delgrupo hemo) y VA (a través de Trp164) (a) y ciclo catalítico propuesto para la VP (b) (Ruiz-Dueñas y Martínez 2010, Pérez-Boada et al. 2005).(b)Los POMs son catalizadores de oxidación conocidos por sus síntesisorgánicas homogéneas y heterogéneas y se han sugerido como alternativas a losreactivos de blanqueo basados en cloro y al blanqueo convencional alcalino conoxígeno (Gamelas et al. 2007). Se utilizan en procesos de deslignificaciónoxidativa con oxígeno, cuyo objetivo principal es la oxidación selectiva de lalignina residual, pudiendo ser regenerados y reutilizados en el proceso dedeslignificación. Se han intentado regenerar POMs por reoxidación con lacasas,pero los resultados no fueron muy esperanzadores para su aplicación industrialdebido a los elevados tiempos de reoxidación, que además no era completa(Gamelas et al. 2007, Gaspar et al. 2007, Tavares et al. 2004, Gamelas et al.2008). En esta Tesis se han utilizado por primera vez peroxidasas para lareoxidación de POMs. En la Figura 17 se puede observar el ciclo catalíticopropuesto para la oxidación de la lignina por el POM así como su reoxidaciónpor la VP. Los POMs se caracterizan por ser aniones que se pueden visualizarestructuralmente como conjuntos agregados metal-oxígeno (Pope 1983). Launidad básica y más común, los octaedros, está formada por un metal rodeadode seis oxígenos (MO 6 ) (Weinstock et al. 1997). Además de M y O, otroselementos que usualmente son designados por X, pueden formar parte de laestructura de los POMs. Los POMs con estructura de Keggin son los másimportantes y los más estudiados actualmente ya que son más estables yfácilmente disponibles (Gamelas et al. 2003). El anión de Keggin estácompuesto por un tetraedro central XO 4 rodeado por 12 octaedros MO 6 , metaloxígeno,compartiendo aristas y vértices. Los octaedros se encuentran en cuatrogrupos M 3 O 13 , que comparten los átomos de oxígeno de los vértices formando elPOM con la fórmula [XM 12 O 40 ] m- , en que XM 12 es la fórmula abreviada.32

1. IntroducciónMn 3+POM oxLigninaH 2 O 2 H 2 OCompuesto 1(enzima activa)H 2 OPOM redMn 2+POM red POM LigninaoxMn 2+Lignina oxMn 3+ Lignina oxCompuesto 2(a)(b)Figura 17. Representación poliédrica de un polioxometalato con estructura de Keggin (a) yciclo catalítico propuesto para la oxidación de la lignina y reoxidación del POM por la VP enlos ensayos de deslignificación realizados durante esta Tesis (b).1.5.2. Degradación enzimática de lípidos: Control del pitchEl control enzimático del pitch en pastas mecánicas de coníferas mediante eluso de lipasas, se puso en práctica como una operación rutinaria en laproducción industrial de papel a principios de los años 90 (Hata et al. 1996) yfue el primer caso en que una enzima se aplicó con éxito en el proceso deproducción papelero. Las lipasas (Figura 18a) son un grupo de hidrolasasproducidas por una gran variedad de organismos. Los tratamientos de pasta conlipasas se iniciaron en Japón con una enzima de Candida cylindracea (Irie 1990)y se continuaron en diversas pruebas de fábrica utilizando una lipasa mejorada ycomercializada por Novo Nordisk (actualmente Novozymes) bajo el nombrecomercial de Resinase® (Matsukura et al. 1990, Fujita et al. 1991, 1992,Gutiérrez et al. 2001), que es capaz de hidrolizar aproximadamente el 95% delos triglicéridos presentes en una pasta mecánica de pino. A lo largo de los añossiguientes, varias fábricas en Japón y China han introducido esta tecnología delcontrol del pitch basada en lipasas en pasteados mecánicos. Las lipasas actúansobre los glicéridos pero no sobre otros extraíbles lipídicos. Teniendo en cuentaque los triglicéridos se hidrolizan con facilidad en el pasteado alcalino (kraft ysosa), las lipasas no son de interés para el control del pitch en estos procesos, asícomo cuando otros compuestos como esteroles libres y conjugados, alcoholesgrasos y alcanos, son los responsables del pitch (del Río et al. 1998, 1999, 2000,Gutiérrez y del Río 2005). Otras enzimas de tipo hidrolasa estudiadas son lasesterol esterasas, que también podrían ser efectivas para el control del pitch yaque los ésteres de esteroles son a menudo causa de pitch, debido a su33

1. Introducciónpegajosidad y resistencia a la cocción alcalina. Sin embargo, estas enzimasliberan esteroles libres que son tan problemáticos como sus ésteres.Por otro lado, las lacasas constituyen un grupo de enzimas oxidativas queha sido objeto de gran interés en el desarrollo de tecnologías respetuosas con elmedio ambiente (Mayer y Staples 2002). Como se ha comentado anteriormente,la acción directa de las lacasas en principio está restringida a las unidadesfenólicas, pero en presencia de mediadores redox (Bourbonnais y Paice 1990,Call 1994) amplían la acción de la lacasa a sustratos no fenólicos, lo queaumenta su potencial en la degradación de lignina y de otros compuestosaromáticos. Recientemente, se ha mostrado la gran eficacia del sistema lacasamediadoren la eliminación de extraíbles lipofílicos de pastas de coníferas,frondosas así como de fibras no madereras (Gutiérrez et al. 2006a, 2006c,2006b, 2007, 2009, Molina et al. 2008).Finalmente, hay que mencionar que se ha sugerido recientemente el usode una lipoxigenasa (de soja) para el control del pitch en pasta termomecánicade coníferas (Zhang et al. 2007). Las lipoxigenasas (Figura 18b) son una clasede dioxigenasas que contienen hierro (no en forma hemo) y catalizan laoxigenación de ácidos grasos insaturados y sus ésteres. El descubrimientoreciente de una lipoxigenasa fúngica (que contiene manganeso) ha revelado laexistencia de otro tipo de lipoxigenasas con una habilidad única para oxidarácidos grasos (Hamberg et al. 1998; Su and Oliw 1998). Esta lipoxigenasa se haevaluado en la Tesis para la eliminación de lípidos y lignina.(a)(b)Figura 18. Estructura de una lipasa (a) y de una lipoxigenasa (b).34

1. Introducción35

2. ObjetivosOBJETIVOSEn esta Tesis se aborda el estudio exhaustivo de la composición química delos principales constituyentes de diversos cultivos lignocelulósicos utilizadoscomo materia prima para la fabricación de pasta de papel, así como de laevolución de los mismos durante el proceso de cocción y blanqueo. Estosestudios tienen por objeto obtener un mejor aprovechamiento industrial de estasfibras, que ayudará a optimizar los procesos de cocción y blanqueo utilizandotecnologías menos contaminantes, incluyendo la biotecnología.Los objetivos específicos de esta Tesis son los siguientes:- Realizar una caracterización química de los diferentes cultivoslignocelulósicos seleccionados, poniendo especial énfasis en lacomposición de lípidos, lignina y hemicelulosas.- Estudiar la modificación estructural de los constituyentes orgánicos delos diferentes cultivos lignocelulósicos durante los procesos de coccióny blanqueo.- Estudiar y desarrollar diferentes aplicaciones biotecnológicas quepermitan degradar tanto la lignina residual como los compuestosextraíbles lipofílicos presentes en las pastas de papel mediantetecnologías menos contaminantes.37

2. Objetivos38

392. Objetivos

3. Material y MétodosMATERIAL Y MÉTODOS3.1. MATERIALESEn esta Tesis se ha estudiado la composición química de fibras de diversoscultivos agrícolas utilizadas para la fabricación de pastas de celulosa de altacalidad y el comportamiento de sus componentes a lo largo del proceso defabricación de dichas pastas. Entre los materiales estudiados se encuentran fibrasdel tallo de varias plantas anuales, tales como lino, kenaf, cáñamo y yute; yfibras procedentes de hojas de sisal, abacá y curauá. También se seleccionaronpastas crudas y pastas blanqueadas (TCF y ECF). Tanto las muestrascorrespondientes a las materias primas como sus pastas fueron suministradas porla empresa Celulosa de Levante S.A., CELESA (Tortosa, Tarragona). Por otrolado, también se estudiaron otras fibras de potencial aplicación en el sectorprocedentes de tallos de caña común, así como fibras de residuos de poda deárboles de tagasaste, que fueron suministradas por la Universidad de Huelva.Finalmente, para la realización de los diversos tratamientos biotecnológicos seutilizaron pastas de eucalipto y de lino, suministradas por las empresas ENCE(Pontevedra) y CELESA, respectivamente.3.1.1. Cultivos lignocelulósicosLinoEl lino (Linum usitatissimum) (Figura 19) es una planta herbáceadicotiledónea de la familia de las Lináceas, originaria de Asia que se cultiva porel aceite de su semilla y por las fibras de sus tallos. Se cultiva principalmente enregiones frías y templadas como Europa, Asia, Australia, Argentina y Brasil.Figura 19. Planta de lino (izquierda) y morfología de sus fibras elementales (derecha) (GarcíaHortal 2007).41

3. Material y MétodosDe las 150 especies del género Linum, sólo la especie L. usitatissimumproduce fibras útiles comercialmente. Las plantas de lino textil alcanzan unaaltura de 0,9-1,25 m y un diámetro de 0,25-0,5 cm. Los haces fibrosos se extraenfácilmente por un procedimiento denominado enriado, que consiste en unproceso hidrolítico en el que enzimas de hongos y bacterias disuelven laspectinas que mantienen adheridas las fibras, proporcionando unas fibras muypuras y de alta calidad que tienen una longitud de 30 a 90 cm. La fibra elementaltiene una longitud comprendida entre 10 y 55 mm y un diámetro de 12 a 30 m,estando entre las más altas de todas las fibras utilizadas en la industria papelera,siendo excedida sólo por la fibra de algodón (García Hortal 2007).La pasta procedente de fibras de lino es ideal para la producción de papelesdelgados a los que se exige una gran resistencia, papeles densos y permanentes,tales como el papel para cigarrillos, papeles muselina, papel biblia y papelespara imprimir de bajo gramaje. Debido a su pureza y resistencia, a su capacidadpara soportar un refinado intenso y por consiguiente dar una pastaextremadamente porosa, es ideal para bolsas de té, papeles para registro, papelesde seguridad y papel moneda.CáñamoEl cáñamo (Cannabis sativa) (Figura 20) es una planta herbácea dicotiledóneade la familia de las Cannabináceas, originaria de Asia central que se cultiva porel aceite y la proteína de su semilla así como por las fibras de sus tallos. Secultiva principalmente en los países de la antigua URSS y en China, pero seencuentra también en la India, países de Europa Central, Paquistán, Turquía,Italia y Colombia. Es una planta que se acomoda a todos los climas, muycompetitiva con las malas hierbas, por lo que no exige grandes inversiones deherbicidas (Struik et al. 2000).Figura 20. Planta de cáñamo (izquierda) y morfología de sus fibras elementales (derecha)(cedida por García Hortal).42

3. Material y MétodosLas plantas de cáñamo presentan tallos que pueden alcanzar alturas de 1 a 5 my diámetros entre 5 y 10 mm. Los haces fibrosos son más largos que los de lino,de 100-300 cm, más rígidos y gruesos. La fibra elemental tiene característicasmorfológicas similares a la del lino, aunque no son tan uniformes, son menostransparentes y con numerosos nudos. Las fibras secundarias, más cortas, sonmás delgadas y más lignificadas (García Hortal 2007).La pasta procedente de cáñamo, al igual que la pasta de lino, es ideal para laproducción de papeles especiales que requieren una fibra fuerte, tales como elpapel para cigarrillos, papel biblia, filtros de café, bolsitas de té, papel aislante ypañales.KenafEl kenaf (Hibiscus cannabinus) (Figura 21) es una planta herbáceadicotiledónea de la familia de las Malváceas, probablemente originaria deÁfrica. Se cultiva en regiones tropicales y subtropicales de la India, Sudeste deAsia y América Central. Es una planta de crecimiento rápido, que se estádesarrollando como fuente de fibras para la fabricación de papel.Las plantas de kenaf crecen en delgadas cañas de hasta 6 m de altura y 4 cmde diámetro. En el tallo, al igual que en el lino y cáñamo, se distinguen dosregiones distintas. Las fibras de la corteza exterior (bast), que constituyealrededor de 30-40% del tallo, son moderadamente largas, mientras que lasfibras del núcleo (core) son más cortas. La longitud de las fibras liberianas es de2 a 6 mm, sin embargo, cabe destacar que las fibras liberianas de las plantastardías son más cortas que las de las plantaciones normales, ya que elcrecimiento vegetativo de esta planta está influenciado por la duración del día(García Hortal 2007, Pande y Roy 1998).Figura 21. Planta de kenaf (izquierda) y morfología de sus fibras elementales (derecha)(García Hortal 2007).43

3. Material y MétodosSe pueden obtener pastas de kenaf tanto de la corteza exterior como delnúcleo, siendo las fibras largas de la corteza exterior especialmente adecuadaspara la fabricación de papeles de calidad especial. Las pastas de kenaf sonideales para papel de prensa, impresión y escritura, y se pueden usar en lamayoría de calidades de papel.YuteEl yute (Corchorus capsularis) (Figura 22) es una planta herbáceadicotiledónea de la familia de las Tiliáceas, que crece principalmente en climashúmedos y cálidos de Paquistán, India, Bangladesh, Brasil y otros paísestropicales. Sus haces fibrosos, que tienen una longitud de 20 a 50 cm ycontienen de 10 a 30 fibras elementales, se usan principalmente para laproducción de sacos, tapices, cuerdas, textiles y materiales para embalaje.Las plantas de yute presentan tallos que pueden alcanzar alturas de 2,5-3 m y10-20 mm de diámetro. La fibra elemental tiene una longitud variable de 1,5 a 5mm, el menos elevado de las otras fibras liberianas mencionadas, y un ancho de10 a 25 m. Los tallos son enriados en estanques de agua, donde se liberan loshaces de fibras liberianas de la corteza y la porción leñosa de la planta (GarcíaHortal 2007, Han 1998).Dureza y durabilidad son las principales características aportadas por lasfibras de yute en mezclas con pastas kraft. Las pastas mecánicas o poco cocidasde yute, debido a la mayor lignificación de sus haces fibrosos respecto a los dellino y cáñamo, se usan para cartones, papeles de estraza y de embalaje, y laspastas químicas y blanqueadas se usan para papeles finos como bolsas de té ypapel de fumar.Figura 22. Planta de yute (izquierda) y morfología de sus fibras elementales (derecha)(García Hortal 2007).44

3. Material y MétodosSisalEl sisal (Agave sisalana) (Figura 23) es una planta robusta de climastropicales originaria de América Central y México, cultivada actualmente enBrasil, Venezuela, Tanzania, Kenia, Mozambique, Angola, Madagascar y otraszonas tropicales. Es una planta monocotiledónea de la familia de las Agaváceasy sus fibras, denominadas fibras duras, más rígidas y bastas que las fibrasliberianas, se han utilizado durante mucho tiempo en las industrias textil y decordelería.La planta de sisal crece hasta alturas de 2 m, con un tronco corto de 15-23 cmde diámetro. Las fibras se extraen de las hojas, que tienen entre 100 y 150, trasser cortadas y descortezadas. El descortezado se debe hacer tan pronto como seaposible una vez que se cortan las fibras, para reducir el deterioro de las mismas.El espesor, longitud y resistencia de la fibra depende de la madurez de la hoja yde su posición a lo largo de ésta, ya que las hojas más maduras contienen lasfibras más largas y bastas, y las fibras más gruesas se hallan en el extremoterminal de la hoja.La hoja madura del sisal mide 1-2 m de largo, 10-15 cm de ancho y 6 mm deespesor (en el centro) y las fibras se disponen longitudinalmente en la hoja. Loshaces fibrosos tienen longitudes de 60-150 cm, y la fibra elemental tiene unalongitud de 1-8 mm y un ancho de 8-40 m, (García Hortal 2007, McDougall etal. 1993).Las fibras del sisal se aplican fundamentalmente en la fabricación de cuerdasy papeles especiales como bolsas de té, papeles dieléctricos y papel de filtro,debido, sobre todo, a la alta porosidad de sus pastas. Por otra parte, también seutilizan como refuerzo para los papeles delgados (Moore 1996)Figura 23. Plantas de sisal (izquierda) y morfología de sus fibras elementales (derecha)(García Hortal 2007).45

3. Material y MétodosAbacáEl abacá (Musa textilis) (Figura 24) es una planta perenne nativa de Filipinasy que se cultiva actualmente también en Indonesia y América tropical. Es unaplanta monocotiledónea que pertenece a la familia de las Musáceas y sus fibrastambién están clasificadas como fibras duras.La planta se asemeja a la del platanero, pero con hojas más pequeñas y frutosno comestibles. Los tallos alcanzan alturas de 3 a 7,5 m y diámetros de 12 a 30cm y consisten en un corazón central envuelto por vainas foliares. Las fibrasútiles comercialmente se encuentran en las vainas externas del tallo, que seextraen por descortezado puramente mecánico de las vainas, de las que seseparan los haces por simple rasgadura.Los haces fibrosos son muy largos, de hasta 2 m y la longitud de la fibraelemental varía de 2,5 a 12 mm según el espesor y la posición en el tallo de lavaina madre, donde las vainas más externas tienen fibras más cortas, másgruesas y de color más oscuro, por lo que son las de menor calidad (GarcíaHortal 2007, Moore 1996).Los papeles producidos por las fibras de abacá son altamente porosos, por loque son ideales para filtros y envolturas de embutidos. Se considera unaexcelente materia prima para papeles de alta calidad, como papel moneda,pañales, servilletas, papel tisú, accesorios para hospitales, etc. El abacá deprimera calidad también se emplea mezclado con algodón o pasta de madera enla fabricación de papeles superfinos, papel para imprimir de bajo gramaje, deregistro, moneda y seguridad y, sobre todo, papel para filtro poroso de uso enlaboratorio o industrial.Figura 24. Planta de abacá (izquierda) y morfología de sus fibras elementales (derecha)(cedida por García Hortal).46

3. Material y MétodosCurauáEl curauá (Ananás erectifolius) es una planta herbácea de fruto no comestible(Figura 25a) perteneciente a la familia de las Bromeliáceas, de origen americanoy hábitat tropical, muy común en el Amazonas. Tiene un tallo tan corto queparece carecer de él y con hojas rígidas. Normalmente produce entre 20 y 24hojas, proporcionando aproximadamente 2 kg de fibra. En la última década, haganado reconocimiento comercial como material para composites en la industriadel automóvil (Leao et al. 1998, Silva et al. 2001). La fibra del curauá tambiénse ha propuesto como una materia prima alternativa para la producción de pastasquímicas en Brasil.Caña comúnLa caña común (Arundo donax) es una planta monocotiledónea perenne,perteneciente a la familia de las Poáceas que parece ser originaria de Asia y queha colonizado el área del Mediterráneo, en países como Portugal y España. Esconsiderada una de las mayores gramíneas, con una estructura tubularsegmentada semejante al bambú (Figura 25b), con alturas entre 2 y 8 m (Seca etal., 2000). Debido a la fácil adaptabilidad de esta gramínea a diferentescondiciones ecológicas, a la elevada productividad de biomasa y capacidad decultura intensiva, es una de las especies no madereras más atractivas comofuente alternativa de fibras para la industria de pasta de papel (Shatalov yPereira 2002).TagasasteEl tagasaste (Chamaecytisus proliferus spp. palmensis) es un arbusto robustode crecimiento rápido perteneciente a la familia de las Fabáceas. Es nativo de lasIslas Canarias y se cultiva en Australia, Nueva Zelanda y otros países. Debido asu alto contenido proteico, se usa como alimento para el ganado y también comocultivo fijador del nitrógeno para mejorar la fertilidad del suelo. Con el fin defomentar la formación de tallos múltiples, el arbusto debe ser podado conregularidad, lo que conduce a una alta acumulación de residuos de poda. Estosresiduos se han evaluado recientemente como materia prima alternativa para laproducción de pasta de celulosa (Díaz et al. 2004, López et al. 2004, Jiménez etal. 2006, 2007, García et al. 2008). En la Figura 25c se muestra una fotografíade un arbusto de tagasaste.47

3. Material y MétodosacbFigura 25. Diversas plantas estudiadas como materias primas potenciales para la producciónde pasta de celulosa: (a) curauá, (b) caña común y (c) tagasaste.3.1.2. Pastas de papelPastas de fibras no madererasSe han estudiado pastas de lino, cáñamo, sisal y abacá, tanto crudas (procesode cocción sosa-AQ) como blanqueadas (procesos de blanqueo TCF y ECF)suministradas por CELESA. Las pastas crudas se obtuvieron por una cocciónsosa-AQ que emplea hidróxido de sodio y antraquinona (hasta 0,05%) comoagentes de cocción, durante 2-4 h a una temperatura de 160-170 ºC.Las pastas blanqueadas se obtuvieron tras secuencias de blanqueo TCF yECF. La secuencia de blanqueo TCF (Q-Po), incluyó una etapa quelato (Q),seguida de una etapa con peróxido de hidrógeno con oxígeno presurizado (Po).La etapa de quelato se lleva a cabo para capturar los iones metálicos y evitar quedichos iones destruyan el peróxido de hidrógeno. La secuencia de blanqueo ECFusada (D-Po) incluyó una etapa de dióxido de cloro (D), seguida por una etapade peróxido de hidrógeno con oxígeno presurizado (Po).Pastas de fibras madererasPara la realización de los diferentes tratamientos biotecnológicos se usó pastakraft cruda (no blanqueada) de eucalipto (E. globulus) suministrada por ENCE.48

3. Material y Métodos3.1.3. Enzimas y mediadoresEn esta Tesis se han estudiado dos aplicaciones biotecnológicas: (i) enzimasde tipo lipoxigenasa para la degradación de lípidos y lignina en pastas kraft deeucalipto y pastas sosa-AQ de lino, y (ii) un sistema compuesto de unpolioxometalato y una enzima de tipo peroxidasa para la degradación de ligninaen pastas kraft de eucalipto.LipoxigenasasLa lipoxigenasa utilizada se obtuvo del hongo ascomiceto Gaeumannomycesgraminis y fue suministrada por la empresa Novozymes (Bagsvaerd,Dinamarca). La unidad de actividad de esta enzima se define como la cantidadde enzima que da lugar a un aumento en la Absorbancia a 234 nm, de 0,001 porminuto a pH 7 y 30ºC, cuando se usa el ácido linoleico como sustrato (volumende reacción = 1,0 mL y camino óptico = 1 cm). Los tratamientos enzimáticoscon lipoxigenasa incluyeron reacciones con varios lípidos modelo, como alcanos(nonacosano), alcoholes (octacosanol), ácidos grasos (ácido linoleico), esteroleslibres (sitosterol) y ésteres de esteroles (colesteril linoleato), representativos delos extraíbles lipofílicos de las pastas estudiadas. El sitosterol fue suministradopor Calbiochem y los demás compuestos por Sigma-Aldrich.PeroxidasasSe utilizó la peroxidasa versátil del hongo basidiomiceto Pleurotus eryngii(VP), producida en el Centro de Investigaciones Biológicas (CIB, CSIC,Madrid) como se describe a continuación.La cepa de E. coli se cultivó en matraces de 1 L con 500 mL de medio TB(Terrific Broth) y 100 μg/mL de ampicilina. El medio TB consiste en un mediotamponado para el crecimiento de la cepa de E. coli transformada con elplásmido portador del gen que codifica la VP de P. eryngii. Se incubó a 37ºC y200 rpm durante 3 horas. Se indujo la expresión añadiendo isopropil tio--Dgalactopiranósido(IPTG) a una concentración final de 1 mM y se continuó conla incubación durante 4 horas más. El “pellet” bacteriano, que contiene laproteína recombinante en forma de cuerpos de inclusión, se obtuvo porcentrifugación, se resuspendió en tampón de lisis (tampón con Tris-HCl pH 8,050 mM, EDTA pH 8,0 10 mM y DTT 5 mM), se le añadió lisozima a unaconcentración final de 2 mg/mL y se incubó durante 1 hora en hielo. Se leañadió 0,1 mg/mL DNAasa, se sonicó en 3 ciclos de 1 minuto a 20.000 Hz defrecuencia y se centrifugó 30 min a 12.500 rpm para favorecer la precipitaciónde los cuerpos de inclusión.49

3. Material y MétodosUna vez lavados los cuerpos de inclusión con una solución de Tris-HCl pH8,0 20 mM, EDTA pH 8,0 1 mM y DTT 5 mM, se resuspendieron en el mínimovolumen posible de la misma solución de lavado, se homogenizaron ycentrifugaron. Se resuspendieron de nuevo los cuerpos de inclusión en elmínimo volumen de solución desnaturalizante (solución de Tris-HCl pH 8,0 50mM, EDTA pH 8,0 1 mM, DTT 5 mM y urea 8 M), se incubaron a temperaturaambiente durante 15 min y se centrifugaron para eliminar los restos no disueltos.El sobrenadante, con la proteína completamente desplegada, se diluyó 5 vecescon una solución de Tris-HCl pH 8,0 50 mM, EDTA pH 8,0 1 mM y DTT 1mM, para llegar a una concentración final de urea de 1,6 M y de 1-2 mg/mL deproteína. Esta solución se añadió a la mezcla de replegado (CaCl 2 5 mM,glutatión oxidado 0,5 mM, Tris-HCl pH 9,5 50 mM y hemina 20 μM) en unaproporción 1:10 para obtener una concentración final de urea de 0,16 M y seincubó en oscuridad a temperatura ambiente durante 16 horas.La mezcla de replegado con la enzima activada se concentró por filtracióntangencial hasta 80-100 mL, a través de una membrana de 3 KDa de tamaño deporo (Membrane Casette de Filtron) con una bomba peristáltica (Masterflexmodelo 7518-02), y posteriormente por ultrafiltración con membrana de 3-10KDa de tamaño de poro en un sistema Amicon® de Millipore hasta 20-40 mL.Se dializó la mezcla concentrada en una solución tampón acetato (acetato 20mM, pH 4,3 y CaCl 2 1 mM) y el material insoluble precipitado se eliminó porcentrifugación. Se dializó de nuevo en solución tampón tartrato (tartrato 10 mM,pH 5,5 y CaCl 2 1 mM) y se purificó por cromatografía líquida de alta resolución(HPLC) en un equipo Äkta de Amersham Pharmacia Biotech de intercambioaniónico previamente equilibrada con el mismo tampón. La proteína se eluyócon un gradiente de NaCl de 0 a 0,3 M en tampón tartrato (tartrato 10 mM, pH5,5, CaCl 2 1 mM) con un flujo de 2 mL/min. Las fracciones obtenidas sedializaron con tartrato 10 mM a pH 5,0 y se realizó el espectro UV-VIS(espectrofotómetro Shimazdu UV-160) para verificar la correcta incorporacióndel hemo durante el proceso de replegado. La concentración de la enzima purase determinó a partir de su coeficiente de extinción molar ( 406 = 150.000 M -1 cm -1 )(Ruiz-Dueñas et al. 1999b). La enzima se congeló en nitrógeno líquido y seconservó a -80ºC hasta su utilización.PolioxometalatosEl POM utilizado fue [SiW 11 Mn III (H 2 O)O 39 ] 5- denominado SiW 11 Mn III enforma simplificada. La solución acuosa del POM se preparó a partir de-K 8 [SiW 11 O 39 ]•13H 2 O, KMnO 4 y Mn(CH 3 COO) 2 •4H 2 O. Se disolvieron 9,6 gde K 8 [SiW 11 O 39 ]•13H 2 O en 13 mL de agua, a 95ºC. En vasos separados sedisolvieron, a temperatura ambiente, 0,095 g de KMnO 4 en 10 mL de HCl0,6mM y 10 mL de agua y 0,6 g de Mn(CH 3 COO) 2 •4H 2 O en 10 mL de agua. Al50

3. Material y Métodosvaso conteniendo K 8 [SiW 11 O 39 ]•13H 2 O a 95ºC se le adicionó poco a poco yconsecutivamente las disoluciones de los otros dos vasos y se dejó unos 25minutos a 95ºC. Se guardó la disolución preparada hasta el día siguiente parafiltrarla y diluirla para 100 mL de modo que la concentración final fuera deaproximadamente 30 mM.3.2. MÉTODOS ANALÍTICOS3.2.1. Aislamiento y análisis de los compuestos lipofílicos de las fibras ypastasEl análisis de los compuestos extraíbles lipofílicos de las fibras y pastasrequirió su aislamiento previo. Dichos compuestos se extrajeron con acetona enun extractor de tipo Soxhlet durante 8 horas. A continuación se evaporó eldisolvente a sequedad en un rotavapor y la cantidad de extracto se determinó porgravimetría. Los extractos lipofílicos obtenidos se redisolvieron en CHCl 3 parasu posterior análisis por cromatografía de gases (GC) y cromatografía degases/espectrometría de masas (GC/MS), descritos más adelante.Fraccionamiento de los compuestos extraíbles lipofílicos mediante SPEPara una caracterización más detallada de los compuestos presentes en losextractos lipofílicos, se procedió a su aislamiento y purificación mediante SPE(extacción en fase sólida) según un método previamente descrito (Gutiérrez etal. 1998, 2004), tal como se muestra en la Figura 26.Los extractos lipídicos se fraccionaron en cartuchos (500 mg) deaminopropilo (Waters, Millipore). Los extractos secos (5-10 mg) seresuspendieron en un volumen mínimo (

3. Material y MétodosEsterolesHidrocarburosesteroidalesEscualenoÁcidos grasosÉsteres de esterolesTriglicéridos1020 30 minHexano4 mLExtracto lipídico total enHexano /CHCl 3 (4:1) 0.5 mLHexanoHexano /CHCl 38 mL(5:1) 6mLCHCl 310 mLÉter/AcOH(98:2) 10 mLFase aminopropiloAcondicionamientoEscualenoÉsteres deHidrocarb.esterolesesteroidalesABCDCerasTriglicéridosEsteroles10 20 30 min10 20 30 minC 16 C 18:2C 18:1Ácidos grasos10 20 30 minC C 2618 C 22C C 24 28 C 30C 2010 20 30 minFigura 26. Esquema del fraccionamiento de un extracto lipídico por SPE (Gutiérrez et al.2004).52

3. Material y MétodosMétodos de derivatización de los compuestos extraíbles lipofílicosPara el análisis por GC y GC/MS es esencial que los compuestos existentes enla muestra sean suficientemente volátiles, por lo que es necesario recurrir amétodos de derivatización cuando los compuestos a analizar no son volátiles,esto es, métodos de conversión de ciertos compuestos en otros que seancompatibles con el método analítico de GC/MS. Las técnicas empleadas en estaTesis incluyeron la metilación de grupos carboxilo y la silanización de gruposhidroxilo.La metilación de los grupos carboxílicos de ácidos grasos libres,hidroxiácidos y grupos fenólicos se realizó con (trimetilsilil)diazometano(TMSD) suministrado por Sigma-Aldrich. Para ello, una vez seca la muestra, seañadió 100 l de metanol y 50 l de una solución de TMSD 2,0 M en hexano yse mantuvo 20 min en el baño de ultrasonidos. A continuación se secó connitrógeno, para resuspenderla en CHCl 3 y analizarla por GC/MS.La silanización de los grupos hidroxilo de alcoholes, esteroles, etc, se realizócon N,O-bis-(trimetilsilil)-trifluoroacetamida (BSTFA) suministrado por Sigma-Aldrich. Para ello, una vez seca la muestra, se añadió 0,2 mL de BSTFA y 0,1mL de piridina. A continuación se calentó a 70ºC durante 2 h y se secó connitrógeno. Posteriormente, se redisolvió en CHCl 3 para analizarla por GC/MS.Análisis de los extractos lipofílicos mediante GC y GC/MSPara el análisis de los compuestos extraíbles lipofílicos por GC y GC/MS, lascaracterísticas de las columnas cromatográficas utilizadas fueron las adecuadaspara separar e identificar los compuestos de alto peso molecular como ceras,ésteres de esteroles, triglicéridos, etc. Previamente se habían realizado estudiossobre procedimientos para el análisis de los extractos lipofílicos de maderas(Gutiérrez et al. 1998a, 2004) por GC y GC/MS en donde se usaron diversascolumnas de diferente longitud y diferentes programas de temperatura. En estosestudios, las columnas capilares seleccionadas para el análisis de lípidos por GCfueron de longitud corta (5 m) ya que proporcionan una conveniente elución yseparación de lípidos de alto peso molecular en un corto período de tiempo (20min). Columnas menores de 5 m no son convenientes ya que no proporcionan laresolución necesaria para análisis cuantitativos. En el caso de los análisis porGC/MS, los cromatogramas obtenidos tienen que ser reproducibles con losobtenidos por GC usando columnas capilares de 5 m. No obstante, en el sistemaGC/MS, debido a las condiciones de alto vacío a las que opera, no se puedenusar columnas tan cortas, por lo que se usaron columnas de 10-15 m. Estalongitud de columna es apropiada para el análisis de lípidos de alto peso53

3. Material y Métodosmolecular por GC/MS proporcionando resultados en un período de tiempo corto(30 min).Los análisis cromatográficos de los extractos lipofílicos, tanto de las muestrasderivatizadas como sin derivatizar, se llevaron a cabo en un cromatógrafo degases Agilent 6890N equipado con un detector de ionización de llama (FID) yuna columna capilar corta de sílice fundida (DB-5HT, J&W; 5 m x 0,25 mm IDy 0,1 m de espesor de película). El programa de calentamiento del hornocomenzó a 100°C (1 min), seguido de un incremento de temperatura hasta350°C (3 min) a 15ºC/min. Las temperaturas del inyector y del detector semantuvieron a 300°C y 350°C, respectivamente. El gas portador que se utilizófue Helio y la inyección se realizó en modo splitless.El análisis mediante GC/MS se llevó a cabo en un cromatógrafo de gasesVarian 3800 acoplado a un detector de trampa de iones (ITD, Varian 4000),usando una columna capilar de sílice fundida (DB-5HT, J&W; 12 m x 0,25 mmID, con espesor de película de 0,1 m). El horno se calentó de 120°C (1 min) a380°C (5 min) a 10ºC/min. La línea de transferencia se mantuvo a 300ºC. Latemperatura del inyector se programó de 120ºC (0,1 min) a 380°C con unarampa de 200ºC/min y manteniéndose hasta el final del análisis. El gas portadorutilizado fue Helio. La identidad de cada componente se determinó porcomparación de sus espectros de masas con los espectros existentes en laslibrerías (Wiley y NIST) y con espectros publicados anteriormente, por susfragmentaciones y, cuando fue posible, por comparación con patronessuministrados por Sigma-Aldrich (octadecano, ácido palmítico, sitosterol,colesteril oleato y sitosteril 3-D-glucopiranósido). Los picos cromatográficos secuantificaron a partir de sus áreas en los cromatogramas. Se utilizó una recta decalibrado, realizada con los patrones anteriormente descritos. En todos los casosse obtuvo un coeficiente de correlación mayor de 0,99.3.2.2. Aislamiento y análisis de la lignina de las fibras y pastasDeterminación del contenido en ligninaEl contenido en lignina de las muestras se determinó por el método Klasonsegún la norma Tappi T222 om-88 (Tappi 2004), con algunas modificaciones.En este método, las muestras molidas de las distintas fibras seleccionadas, libresde compuestos extraíbles, se sometieron a una hidrólisis con H 2 SO 4 al 72%(p/p), a 30ºC durante 1 h. Posteriormente, la solución se diluyó hasta alcanzaruna concentración del 4% en H 2 SO 4 y se autoclavaron (1h a 110ºC). Acontinuación, las muestras se filtraron, guardándose los primeros 100 mL para elposterior análisis de los azúcares libres y el residuo insoluble (lignina Klason) se54

3. Material y Métodoslavó con agua destilada hasta pH neutro y se secó para su cuantificacióngravimétrica.Aislamiento de la lignina de las fibrasLa lignina se extrajo de las muestras, previa eliminación de los compuestoslipofílicos e hidrosolubles de las fibras, según el protocolo desarrollado porBjörkman (1956) que consiste en extraer la lignina de la muestra finamentemolida. El grado de molienda deseado se alcanzó utilizando un molino de bolascentrífugo modelo Retsch S100 durante 100 h. La lignina de las muestrasfinamente molidas se extrajo con dioxano-agua (9:1, utilizando 250 mL por cada10 g de muestra) durante 12 h. Posteriormente, se centrifugó (25 min, 4ºC,11000 rpm) y se recogió el sobrenadante (que contiene la lignina) en un matraz,repitiéndose este proceso dos veces de forma consecutiva, y se secó en rotavapora 40ºC. A continuación, el residuo seco se disolvió en una mezcla de ácidoacético:agua (9:1), añadiéndose 20 mL por cada gramo de residuo seco. Lalignina se precipitó en agua destilada (225 mL por cada gramo de lignina) enconstante agitación y se recogió tras centrifugación. Una vez seco el residuo, setrituró en un mortero de ágata para facilitar su disolución en una mezcla 1,2-dicloroetano-etanol (2:1) y de nuevo se volvió a centrifugar a baja velocidad(5000 rpm durante 5 min) recuperando el sobrenadante, que contiene la lignina.Éste se dispersó gota a gota, sin agitar, en éter dietílico (225 mL es suficientepara 0,5-1 g de lignina), precipitando de nuevo la lignina. Se volvió a centrifugar(5000 rpm durante 5 min) y el residuo se resuspendió en éter de petróleo en elque se dejó durante 12 horas. Finalmente, se centrifugó y se secó mediantecorriente de nitrógeno y se conservó a 4ºC, preservándola de la luz y el aire paraevitar su oxidación hasta el momento del análisis. Con este método se obtuvouna lignina poco degradada y representativa de la lignina nativa de las fibras.Análisis de la lignina mediante Py-GC/MSLa pirólisis es un método degradativo que transforma compuestos complejosno volátiles en una mezcla de fragmentos volátiles por descomposición térmicaen ausencia de oxígeno (Meier y Faix 1992; Fullerton y Franich 1983) que selleva a cabo habitualmente a temperaturas de 400-800ºC. En la pirólisis seproducen roturas de los enlaces por acción del calor, ya que cuando la energíaaplicada a la molécula es mayor que la energía de enlaces específicos ocurre ladisociación de éstos de una forma predecible y reproducible, pudiéndose obtenerinformación sobre la molécula original a través del análisis de los productos dedegradación. Los fragmentos resultantes de la pirólisis se pueden separar por GCe identificar por MS. La Py-GC/MS es un método poderoso para el análisis demateriales lignocelulósicos, especialmente de la lignina. La lignina se piroliza55

3. Material y Métodosproduciendo una mezcla de compuestos fenólicos que resultan de la rotura nosólo de enlaces éter, sino también de ciertos enlaces C-C, reteniendo estosfenoles las características de sustitución del polímero de lignina y siendo posiblepor lo tanto identificar los diferentes componentes de ligninas provenientes deunidades H, G y S. La Py-GC/MS presenta diversas ventajas frente a otrosmétodos degradativos, pues es una técnica analítica rápida que proporcionaresultados en apenas un paso, que necesita poca cantidad de muestra y unasimple preparación de la misma. También presenta ventajas frente a los métodosclásicos de análisis de la lignina, pues no es necesario aislar la lignina de lamuestra, permitiendo su análisis in situ.En la presente Tesis, la pirólisis de las muestras se llevó a cabo en unpirolizador Frontier Laboratories Ltd., a 500ºC durante 10 s. El pirolizadorestaba conectado a un cromatógrafo de gases Agilent 6890 con una columnacapilar HP 5MS (30 m x 0,25 mm ID, y un espesor de película de 0,25 m)acoplado a un espectrómetro de masas Agilent 5973 N. El cromatógrafo seprogramó de 50ºC (1 min) a 100ºC con un incremento de 30ºC/min y de 100 a300ºC con un incremento de 10ºC/min. La temperatura final se mantuvo durante10 min. El gas portador utilizado fue Helio con un flujo controlado de 1mL/min. Para las pirólisis en presencia de hidróxido de tetrametilamonio(TMAH), se adicionó 0,5 μL de TMAH 25% a 100 μg de muestra. Loscompuestos obtenidos mediante Py-GC/MS se identificaron por comparacióncon la literatura (Faix et al. 1990, Ralph y Hatfield 1991) y con los incluidos enlas librerías de espectros de masas Wiley y NIST. Se calcularon las áreasmolares para los productos de la pirólisis (lignina y carbohidratos), senormalizó al 100% y se hizo una media entre las repeticiones de las pirólisis. Ladesviación estándar fue inferior al 5% de la media.Análisis de la lignina mediante DFRCEl método de degradación química de la lignina conocido como DFRC(derivatization followed by reductive cleavage) es un método simple y poderosoque rompe de manera selectiva y eficiente los enlaces éter -O-4 y -O-4presentes en la lignina. Permite el análisis cuantitativo de las unidadesestructurales de la lignina eterificadas y también ofrece información sobre losenlaces carbono-carbono a través del análisis de las estructuras diméricas que seforman.El método DFRC incluye dos pasos fundamentales: (i) la solubilización de lalignina por bromación y acetilación con bromuro de acetilo y (ii) lafragmentación reductora de los enlaces aril éter en la lignina con polvo de zinc.La identificación de los productos resultantes de la degradación (monómeros ydímeros) por GC/MS proporciona información valiosa sobre la estructura de la56

3. Material y Métodoslignina (Lu y Ralph 1997a, 1997b, 1998). Una de las ventajas de este método esque deja intacto el carbono de la cadena lateral de la lignina, por lo que lo hacemuy adecuado para estudiar la presencia de unidades aciladas (con acetatos, p-cumaratos o p-hidroxibenzoatos) en el carbono . Sin embargo, el método deDFRC usa reactivos acetilantes que interfieren en el análisis de los gruposacetatos nativos en la lignina, pero con las modificaciones apropiadas, mediantela sustitución de la acetilación por la propionilación (DFRC’), es posible obteneruna información significativa sobre la presencia de unidades acetiladas en lalignina nativa (Ralph y Lu 1998). En la Figura 27a se puede observar la roturaselectiva de los enlaces éter en el método DFRC y en la Figura 27b lamodificación de este método (DFRC’) para el análisis de ligninas naturalmenteacetiladas.La degradación DFRC se llevó a cabo con 10 mg de lignina aislada que setrató con bromuro de acetilo en ácido acético (8:92) durante 2 h a 50ºC. Despuésde proceder a la eliminación del disolvente en rotavapor, se disolvió en unamezcla de dioxano/ácido acético/agua (5:4:1) con polvo de zinc y se dejó enagitación durante 30-40 min a temperatura ambiente. A continuación se ajustó elpH hasta un valor inferior a 3 por adición de HCl 3% y se pasó la disolución aun embudo de extracción para separar las fases orgánica y acuosa por extracciónlíquido-líquido con diclorometano. A la fase orgánica extraída se le añadiósulfato de sodio anhidro y se filtró, recogiéndose el filtrado para eliminación deldisolvente en rotavapor. El residuo que quedó tras la evaporación, se acetilódurante 1 h con anhídrido acético, diclorometano y piridina. Se evaporó eldisolvente en rotavapor con etanol, se resuspendió en diclorometano y seprocedió a su análisis por CG/MS. En el caso de la DFRC’ el protocolo aplicadofue el mismo pero sustituyendo todos los reactivos acetilantes por reactivos conpropionilo.Los análisis mediante GC/MS se llevaron a cabo en un cromatógrafo de gasesVarian Star 3400 acoplado a un detector de trampa de iones (ITD, Varian Saturn2000), usando una columna capilar de sílice fundida (DB-5HT, J&W; 12 m x0,25 mm ID, con espesor de película de 0,1 m). El horno se calentó de 50°C(0,2 min) a 100°C a 30ºC/min y se elevó a 300ºC a 5ºC/min. El inyector y lalínea de transferencia se mantuvieron a 300ºC. El gas portador utilizado fueHelio. La cuantificación de los monómeros se realizó usando tetracosano comopatrón externo asumiendo factores de respuesta similares a los obtenidos por Luy Ralph (1997).57

3. Material y MétodosHOAcOOAc(a)HOOBrOAcBrZnCH 3 OOOCH 3CH 3 OOOCH 3Ac 2 O/PyCH 3 OOAcOCH 3ROROORHOOBrOAcBrZnCH 3 OOOCH 3CH 3 OOOCH 3Ac 2 O/PyCH 3 OOAcOCH 3HOPropOOProp(b)HOOBrOPropBrZnCH 3 OOOCH 3CH 3 OOOCH 3Prop 2 O/PyCH 3 OOCH 3OPropAcOAcOOAcHOOBrOPropBrZnCH 3 OOOCH 3CH 3 OOOCH 3Prop 2 O/PyCH 3 OOCH 3OPropFigura 27. Rotura selectiva de los enlaces éter: (a) Método DFRC (Lu y Ralph 1997a) y (b)Método DFRC modificado (DFRC’) para el análisis de ligninas naturalmente acetiladas(Ralph y Lu 1998).58

3. Material y MétodosAnálisis de la lignina mediante 2D-NMRLa NMR, tanto de 1 H como de 13 C, ofrece una información detallada de laestructura de la lignina, incluyendo los diferentes tipos de unidades y los enlacesque se establecen entre ellas (Robert 1992). En los últimos años, se hadesarrollado la NMR bidimensional (2D-NMR) y tridimensional (3D-NMR), enlas que se establecen correlaciones 1 H- 13 C, entre otras, que resuelve señales queaparecían solapadas en los espectros unidimensionales (Capanema et al. 2001,Ralph et al. 2001, Liitiä et al. 2003). La 2D-NMR se considera en la actualidadla técnica más potente para el análisis de la estructura de la lignina y a través deella se han podido identificar nuevas sub-estructuras tales como lasdibenzodioxocinas (Karhunen et al. 1995) o las espirodienonas (Zhang yGellerstedt 2001, Zhang et al. 2006).El HSQC (Heteronuclear Single Quantum Correlation) proporcionacorrelaciones a través de acoplamiento escalar a un enlace entre un protón y elheteronúcleo al que está directamente unido. Los espectros HSQC de la ligninapresentan tres regiones bien diferenciadas: región alifática, región alifáticaoxigenada y región aromática (Figura 28). La región alifática oxigenada (Figura29) es la más importante para el estudio de la estructura de la lignina ya que enesta zona se encuentran las correlaciones de la mayoría de los enlaces que tienenlugar entre las distintas unidades estructurales. La región aromática (Figura 30)es la más importante desde el punto de vista de la composición de la lignina, yaque en esta zona aparecen las correlaciones de las distintas unidades H, G y S.0Regiónalifática50Región alifáticaoxigenada C (ppm)Regiónaromática10010.05.0 5 0.0H (ppm) 0ppm (t2)Figura 28. Espectro HSQC de la lignina aislada de sisal donde se pueden observan sus tresregiones características.150150ppm (t159

3. Material y Métodos-OMe-OAc-OH’ (-Oac) (-OH)MeORHO65OO MeO6’5’ 1’ 4’ 2’3’O1234OOMeOMeMeOHO MeOHO O6514O23OMe5’4’6’1’2’3’OMeMeOO546123OMeOO’’ ’OMe2’3’4’1’6’5’OOMeMeOHOOMeO 123645 32456MeO1 MeOO O ´ ´6´5´1´4´O2´3´´OMeOHOMeFigura 29. Región alifática oxigenada del espectro HSQC de la lignina aislada de sisal y suscorrespondientes estructuras.60

3. Material y MétodosS 2,6S’ 2,6S’’ 2,6G 2G 52’G 66'MeOHO651234OSOMeMeOO651234OHOMeS’MeOO65OH1234OOMeS’’HO16 25 34OOMeGMeOOHO3456MeOOMe21 MeOO O ´ ´6´5´1´4´O2´3´651234´OMeOHOMeFigura 30. Región aromática del espectro HSQC de la lignina aislada de sisal y suscorrespondientes estructuras.61

3. Material y MétodosLos espectros de NMR de ligninas se registraron en un espectrómetro BrukerAVANCE 500 MHz, equipado con una sonda triple con gradientes en el eje z, auna temperatura de 298ºK. Se disolvieron 40 mg en 0,75 mL de dimetilsulfóxidodeuterado (DMSO-d 6 ). Los experimentos HSQC se realizaron empleando unamatriz de datos de 256 incrementos en la dimensión indirecta, y adquiriendo1024 puntos en la dimensión de adquisición. La constante de acoplamiento 1 J CHutilizada fue de 140 Hz. La intensidad de las señales en los espectros HSQCdependen del valor de esta constante así como del tiempo de relajación T 2(Zhang y Gellerstedt 2007). Por ello, la integración de las señales se realizó porseparado en cada una de las regiones del espectro, utilizando las señalescorrespondientes a correlaciones C-H químicamente análogas, con constantes deacoplamiento 1 J CH similares. En la región alifática oxigenada, las abundanciasrelativas de las diferentes subestructuras de la lignina se estimaron mediante laintegración de las correlaciones C-H. En la región aromática, lascorrelaciones 1 H- 13 C de las unidades S y G se usaron para estimar las relacionesS/G.3.2.3. Aislamiento y análisis de las hemicelulosas de las fibras y pastasPreparación de la holocelulosa y aislamiento de los xilanosLas holocelulosas se obtuvieron por deslignificación de las fibras y pastascrudas (5,0 g) con ácido peracético 10% (pH 3,5) durante 20 minutos a 85ºC.Una vez ocurrida la deslignificación, la holocelulosa se filtró, se lavó conacetona y agua destilada caliente y se secó para su determinación gravimétrica.Los xilanos se aislaron de las correspondientes holocelulosas (molidaspreviamente) con DMSO a 50ºC durante 24 h y se precipitaron con un exceso de7:2:1 etanol:metanol:agua acidificado con ácido acético a 4ºC durante 3-4 días.El precipitado se recogió tras centrifugación, se lavó con metanol y se secósobre vacío. En el caso de las pastas blanqueadas, los xilanos se aislarondirectamente de dichas pastas sin previo aislamiento de las holocelulosascorrespondientes.Análisis de azúcares neutros tras hidrólisis ácidaLos xilanos aislados se sometieron a una hidrólisis con H 2 SO 4 72% durante 3horas a 20ºC, seguido de otra hidrólisis con H 2 SO 4 4% durante 2,5 horas a100ºC. Los monosacáridos neutros se determinaron como acetatos de alditol porcromatografía de gases (Selvendran et al. 1979).62

3. Material y MétodosEl análisis por cromatografía de gases se realizó en un equipo Varian 3350con un detector FID y columna DB-225 J&W (30 m × 0,25 mm ID y un espesorde película de 0,15 μm). El programa de temperatura se programó de 220ºC (5min) a 230ºC con un incremento de 2ºC/min. La temperatura final se mantuvodurante 5 min. Las temperaturas del inyector y detector fueron de 230ºC,respectivamente. La cuantificación se realizó con ayuda de curvas de calibraciónrealizadas con patrones.Análisis de azúcares neutros y ácidos urónicos tras metanolisis ácidaLa fibra o pasta seca (4,5 mg) se sometió a metanolisis ácida añadiendo 2 mLde una disolución de HCl 2 M en metanol anhidro a 100ºC durante 4 horas(Sundberg et al. 1996). Una vez frío, se adicionó 80 μL de piridina paraneutralizar la disolución ácida y se adicionó 1 mL de una disolución 0,1 mg/mLde sorbitol como patrón interno. Se extrajo 2 mL del sobrenadante, se secó en unrotavapor a 40-50ºC, se resuspendió en 70 μL de piridina y se le añadió 150 μLde hexametildisiloxano y 80 μL de trimetilclorosilano para promover lasililación durante 12 horas a temperatura ambiente.Los análisis de las muestras sililadas se llevaron a cabo en un cromatógrafode gases Hewlet-Packard 5890 equipado con un detector de masas MSD seriesII, usando Helio como gas portador (35 cm/s) y una columna capilar (DB-1J&W; 30 m x 0,32 mm ID y 0,25 m de espesor de película). El horno secalentó de 100°C a 175°C a 4ºC/min y de 175ºC a 290ºC a 12ºC/min. La líneade transferencia se mantuvo a 300ºC. La temperatura del detector (FID) fue de290ºC. La identidad de cada componente se determinó por comparación de susespectros de masas con los espectros existentes en las librerías (Wiley y NIST) ycon espectros publicados anteriormente (Sundberg et al. 1996, Bertaud et al.2002, Bleton et al. 1996).Determinación del peso molecular de los xilanos mediante SECEl peso molecular de los xilanos solubles en Me 2 SO se valoró mediantecromatografía de exclusión molecular. Los xilanos aislados con Me 2 SO sedisolvieron en una disolución al 10% de LiCl en N,N-dimetilacetamida(DMAC) y luego se diluyeron con DMAC hasta una concentración deaproximadamente 0,5% (5 mg/mL). Los análisis por SEC se realizaron encolumnas de PLgel 10μm (Mixed B 300 × 7,5 mm) con una pre-columna PLgel10μm (Polymer Laboratories, UK) usando un sistema PL-GPC 110 (PolymerLaboratories). Las columnas, el inyector y el detector se mantuvieron a 70ºCdurante el análisis. El eluyente fue LiCl en DMAC 0,1 M con un flujocontrolado de 0,9 mL/min. Las columnas se calibraron con patrones (Polymer63

3. Material y MétodosLaboratories) en un rango de 0,8-100 kDa. El volumen de muestra que seinyectó fue de 100 μL.Análisis de la estructura de los xilanos mediante NMRLos xilanos aislados se analizaron tanto por 1 H-NMR como por 2D-NMR.Los espectros 1 H-NMR se obtuvieron en un espectrómetro Bruker AVANCE300. Se utilizó como patrón interno ( 0,00) 3-(trimetilsilil) propionato de sodiod4 . Los experimentos 1 H-NMR se realizaron a 30ºC con un pulso de 90º, untiempo de relajación de 16 s y adquiriendo 400 puntos.Los espectros 2D-NMR se obtuvieron en un espectrómetro Bruker AVANCE300. Los experimentos 2D 1 H- 1 H COSY se realizaron a 50ºC usando unasecuencia COSY patrón (pulso de 90º y un tiempo de relajación de 2 s). Losexperimentos 2D 1 H- 1 H TOCSY (mix= 0.050 s) se realizaron empleando unaanchura espectral de 2185 Hz en ambas dimensiones a 60ºC. El tiempo derelajación fue de 2 s. Se emplearon 128 acumulaciones para cada FID,adquiriendo 1024 puntos en t 1 × 512 en t 2 . Finalmente, los experimentos HSQCse obtuvieron a 50ºC empleando una anchura espectral de 12,000 Hz (F1) y de2000 Hz (F2), una matriz de datos de 2048 × 1024 incrementos y 128acumulaciones. El tiempo de relajación fue de 2 s y la constante deacoplamiento 1 J CH utilizada fue de 148 Hz.Determinación del contenido en ácidos hexenurónicosDurante el proceso kraft, los ácidos 4-O-metil-D-glucurónicos, presentes en lacadena lateral de los xilanos, se convierten en los ácidos insaturadoscorrespondientes (ácidos hexenurónicos, HexA) a través de la pérdida del grupometoxilo. Los HexA interaccionan con los agentes químicos de blanqueo y otrosreactivos disminuyendo la blancura de las pastas. Los HexA interfieren en elmétodo de determinación del índice kappa, ya que reaccionan con elpermanganato de potasio, haciendo que se obtenga un valor más elevado delcontenido de lignina residual que el real (Göran y Liebing 1996).El contenido en HexA se determinó a través del método de la hidrólisis ácida,por el que los ácidos hexenurónicos se convierten selectivamente en derivadosdel furano por hidrólisis en tampón formato de sodio a pH 3,0 (Vuorinen et al.1999). La determinación de la cantidad de HexA, se basa en la cuantificación delos derivados del furano que se forman a través del análisis del espectro deUV/VIS.Se dejaron impregnar aproximadamente 0,75 g de pasta en 0,75 mL detampón formato de sodio 10 mM sobre agitación durante una noche. Esta64

3. Material y Métodosmezcla se transfirió al reactor, se substituyó el aire por nitrógeno, se encendió laagitación mecánica y se llevó la temperatura hasta 110ºC durante 1h. Una vezacabada la reacción se filtró la pasta y se lavó, recogiendo siempre el líquido defiltrado, diluyéndose todo hasta 500 mL. Se leyó entonces el valor de laabsorbancia a 245 nm y 480 nm. Se realizó también un ensayo en blanco.El contenido en HexA existentes en las pastas se determinó mediante lafórmula:C ( A245 A480 )8,7 mSiendo,C = cantidad de HexA en la pasta (meq/Kg)A 245 = Absorbancia a 245 nmA 480 = Absorbancia a 480 nmM = peso de la pasta seca (Kg)8,7 mM -1 cm -1 = coeficiente de extinción molar a 245 nm con relación a losHexA.3.2.4. Otros análisisDeterminación de la fracción hidrosoluble de las fibrasEl porcentaje de compuestos hidrosolubles en las fibras se determinó según lanorma Tappi T 207 om-88 (Tappi 2004). Para ello, los cartuchos de las muestrasextraídas con acetona, una vez secos, se colocaron en matraces con 100 mL deagua destilada y se tuvieron en un baño a 100ºC durante 3 h, al cabo de lascuales el extracto se concentró en rotavapor y se secó a 100ºC para sudeterminación gravimétrica.Determinación del contenido en cenizas de las fibrasEl contenido en cenizas se determinó mediante la norma Tappi 211 om-85(Tappi 2004). Para ello se depositaron 200 mg de cada una de muestras encrisoles de porcelana previamente tarados y se introdujeron en la mufla a 575 ºCdurante 6 h. Para tararlos se limpiaron con HCl y se introdujeron en la mufla a575ºC durante 1 h. Posteriormente se sacaron los crisoles de la mufla y se65

3. Material y Métodospesaron una vez que alcanzaron la temperatura ambiente. Los contenidos encenizas se expresaron como porcentajes de la materia prima inicial.Análisis de metales y otros elementos en las fibrasLas fibras seleccionadas, una vez lavadas y secas, se molieron en un molinode cuchillas y se les realizó una digestión con 4 mL de HNO 3 concentrado por0,5 mg de muestra, dejándolas 15 min en un horno microondas (Jones y Case1990). Posteriormente se filtraron con filtro Whatman del número 2, y serecogieron en un matraz que se enrasó hasta 50 mL. La concentración demetales en la disolución obtenida se determinó por espectrometría de emisiónpor plasma (ICP-OES) en un espectrómetro Termo Jarrel Ash, modelo IRISAdvantages.3.2.5. Tratamientos enzimáticos de las pastasTratamientos con lipoxigenasasLos tratamientos de pastas de eucalipto y lino con lipoxigenasa se realizaroncon 5 g de pasta seca, al 1% de consistencia (peso/peso) en tampón dihidrógenofosfato sódico 100 mM (pH 7). La dosis de enzima fue de 10 mg lipoxigenasa/gde pasta de eucalipto y 20 mg de lipoxigenasa/g de pasta de lino, la temperaturade 30ºC, y el tiempo de reacción de 4 horas. Los tratamientos se realizaron enmatraces de 1L, con burbujeo de oxígeno en un baño térmico con agitación (170rpm). En una etapa posterior, las pastas al 5% de consistencia, se sometieron auna etapa de blanqueo con peróxido, usando H 2 O 2 al 3% (peso/peso) y NaOH1,5% (peso/peso), ambos referidos al peso de la pasta seca, a 90ºC durante 2horas, en unas bolsitas selladas de plástico termorresistente. Los controles parala evaluación de la acción de la lipoxigenasa se trataron bajo las mismascondiciones pero sin enzima.Una vez tratadas las pastas, se extrajeron con acetona en un extractor de tipoSoxhlet durante 6 horas. A continuación se evaporó el disolvente a sequedad enun rotavapor y los extractos lipofílicos obtenidos se redisolvieron en CHCl 3 parasu posterior análisis mediante GC y GC/MS, en las condiciones descritasanteriormente para el análisis de los compuestos lipofílicos. Se realizaronanálisis posteriores de las propiedades de las pastas (blancura ISO, índice kappa,viscosidad intrínseca y ácidos hexenurónicos).También se llevaron a cabo reacciones enzimáticas con compuestos modelo.Para las reacciones con compuestos modelo, se usó 1 mg de cada compuesto yse añadió 0,1 mg de lipoxigenasa, y Tween 20 como dispersante (1% v/v). El66

3. Material y Métodostratamiento se realizó a pH 7, usando tampón dihidrógeno fosfato sódico 100mM, durante 2 horas. Se burbujeó oxígeno dentro de los matraces de reacción, yla reacción se llevó a cabo en un baño con agitación a una velocidad de 100 rpm.En los controles de los experimentos, los lípidos se someten a las mismascondiciones de reacción, sin lipoxigenasa. En una etapa posterior se realizó unafase de peróxido, en la que se añadía a cada matraz de reacción 50 L de H 2 O 2 al30% (p/v) y 37,5 L de NaOH 5N, se taparon los matraces y se colocaron en elbaño térmico de agitación, a 90ºC y 100 rpm, durante 2 horas. Las dispersionesde los lípidos se secaron en rotavapor, se recogieron con cloroformo-metanol(1:1), que se secó luego con nitrógeno, y se redisolvieron en cloroformo para suanálisis mediante GC y GC/MS.Tratamientos con POM y peroxidasa versátilLos ensayos de blanqueo de pasta kraft de eucalipto con POM se llevaron acabo en un reactor PARR modelo 4842 (0,25 L) equipado con un sistema decontrol de temperatura y mecanismo de agitación (Figura 31).Figura 31. Sistema usado para los ensayos de deslignificación.67

3. Material y MétodosPreviamente a la realización de los ensayos de deslignificación, se optimizó lacinética de oxidación del POM reducido (SiW 11 Mn II ) con peroxidasa versátil(VP) para conocer las cantidades a utilizar de peróxido y enzima en losdiferentes ensayos de deslignificación. La reoxidación del POM por la VP fuemonitorizada por espectroscopía de UV/VIS. El color amarillo del POM red ,SiW 11 Mn II , cambia gradualmente durante la oxidación con la VP a un color rosacaracterístico del POM ox , SiW 11 Mn III , (Figura 32a), mostrando una banda detransición d-d* con un máximo de Absorbancia a 490-495 nm (Figura 32b). Serealizaron varias pruebas en tampón acetato 0,1 M, pH 4,5 con objeto demaximizar la oxidación del POM con VP/H 2 O 2 , variando para esto la relaciónH 2 O 2 /POM y POM/VP. Las lecturas se realizaron a un valor de longitud de ondafijo (490 nm) para intervalos de tiempo de 1 minuto, considerando de(SiW 11 Mn III )= 327 y de (SiW 11 Mn II )= 19 en cm -1 mol -1 L. Todas las medidasespectrofotométricas se realizaron en un espectrofotómetro Jasco V-560 UV/Visa temperatura ambiente.Una vez conocidas las condiciones óptimas para la reoxidación del POM, seprocedió a la realización de los diferentes ensayos de deslignificación. Para cadauno de los ensayos, se dejó la pasta (8 g) en agua destilada (600 mL) duranteuna noche en agitación constante. Se filtró, se pasó al reactor y se adicionó 67mL de tampón acetato de sodio 0,2 M pH 4,5 (concentración final de 0,1 M),disolución de POM concentrado (entre 12,5 a 13,5 mL para que la concentraciónfinal de POM en la mezcla fuera de aproximadamente 3 mM) y agua destiladahasta 134 mL de volumen total (considerando todavía la contribución del aguaretenida en la pasta filtrada), para obtener una consistencia final del 6%. Sepresurizó el reactor con pO 2 =5 bar, se encendió la agitación mecánica y se llevóhasta la temperatura de 110ºC. Una vez acabado el ensayo, se filtró la pasta y selavó con agua destilada (POM 1-). En los casos en que los líquidos de filtradoconteniendo el POM fueron reoxidados (-VP-), se adicionó a estos la enzima yel peróxido de hidrógeno en las cantidades necesarias (obtenidas a través de losensayos de optimización descritos anteriormente), manteniendo agitaciónconstante y a temperatura ambiente (para no desnaturalizar la enzima), duranteaproximadamente 20 min (aunque a los seis minutos la reoxidación ya seestimaba como completa). El filtrado conteniendo el POM reoxidado se añadiónuevamente a la pasta filtrada reiniciando una nueva etapa de deslignificación(-POM 2-).Finalmente, todas las pastas deslignificadas se sometieron a una extracciónalcalina con NaOH (-E) 2% durante 1 hora a 70 ºC. Después de las extraccionesalcalinas, se lavaron las pastas con agua destilada, hasta alcanzar pH neutro en ellíquido de filtrado y se dejaron secar a temperatura ambiente durante 4 días. Serealizaron análisis posteriores de las propiedades de las pastas (blancura ISO,índice kappa, viscosidad intrínseca y ácidos hexenurónicos).68

3. Material y Métodos(a)(b)Figura 32. (a) Colores característicos de SiW 11 Mn III (izquierda) y de SiW 11 Mn II (derecha) y(b) Espectros de UV/VIS de disoluciones acuosas de a: SiW 11 Mn II , b-d: diferentes porcentajesde oxidación hasta e: SiW 11 Mn III (la oxidación máxima).Determinación de las propiedades de las pastasDeterminación de la blancura ISOLas medidas de blancura en porcentaje ISO de las pastas fueron realizadas enENCE y UPC (Terrassa) en el caso de los tratamientos con lipoxigenasas y en elInstituto Raíz de Aveiro (Portugal) en el caso de las pastas tratadas con POM yperoxidasa versátil.El método para la determinación de la blancura ISO mide el factor dereflectancia difusa en azul (grado de blancura ISO) de pastas de papel ycartones. El alcance de esta norma está restringido a pastas de papel y cartonesblancos o casi blancos.Determinación del índice kappaEl procedimiento estándar utilizado en la industria para determinar el gradode deslignificación en una pasta química es la determinación del índice kappapor la norma TAPPI T 236cm-85 (Tappi 2006) y consiste en el volumen en mLde una disolución de KMnO 4 0,1 N consumido por 1 g de pasta. La lignina de lapasta reacciona con el permanganato y la cuantificación del permanganatoconsumido se determina por valoración con tiosulfato de sodio.Con ayuda de una batidora de mano, se desintegraron los gramos de pastanecesarios para el ensayo (ver Anexo 1) en 140 mL de agua destilada y se lavóel pie de la batidora con 50 mL de agua. Con agitación constante, se adicionó69

3. Material y Métodosuna mezcla de 25 mL de KMnO 4 0,1 N y 25 mL de H 2 SO 4 0,2 N. Al cabo de 5minutos se midió la temperatura y después de 10 minutos se paró la reacción con5 mL de KI 1,0 N, se colocaron unas gotas de solución indicadora de almidón al0,2% y se valoró el I 2 liberado con una disolución de Na 2 S 2 O 3 0,2 N.Previamente, se realizó siempre un ensayo en blanco que no contenía pasta.El índice kappa se calculó por la expresión:IK p fw10,013(25 t)p ( b a)N0,1Con:IK = índice kappa.p = Volumen de permanganato de potasio 0,1 N consumido en el ensayo(mL).f = factor de corrección para un consumo de 50% de permanganato de potasioy que depende de p (Anexo 2).w = peso de pasta seca (g).b = volumen consumido de tiosulfato de sodio para determinación del blanco(mL).a = volumen consumido de tiosulfato de sodio para determinación de lamuestra (mL).N = normalidad de la disolución de tiosulfato de sodio.t = Temperatura del medio de reacción (ºC).Determinación de la viscosidad intrínsecaLa viscosidad de las pastas está directamente relacionada con el grado depolimerización de las moléculas de celulosa y por lo tanto con la resistencia de70

3. Material y Métodoslas fibras. Se determinó la viscosidad intrínseca de las pastas a través de lanorma SCAN-CM 15:85 (SCAN 1994). Este método permite determinar laviscosidad de las pastas celulósicas solubles en una disolución de cobre (II)-etilendiamina (CED) en un viscosímetro capilar.Se pesó la cantidad de pasta necesaria para el ensayo (ver Anexo 3) y setransfirió a frascos de 61 mL, se adicionó 25 mL de agua destilada y 5 hilos decobre. Se colocaron los frascos a agitar durante aproximadamente 30 minutos enun agitador de brazos. Seguidamente, se adicionó 25 mL de CED 1,0 M para darlugar a la disolución de los polisacáridos. Se completó el volumen del frasco condisolución de CED 0,5 M usando una bureta, se cerraron los frascos para nodejar burbujas de aire y se colocaron de nuevo sobre agitación durante otros 30minutos. Finalmente, se anotó el tiempo de escurrido de 1 mL de pasta disueltaen CED, repitiendo la lectura 3 veces para cada frasco, usando un viscosímetrocapilar a una temperatura controlada de 25ºC.La viscosidad relativa de las pastas fue determinada por la expresión: rel = h×t nCon:h = constante del viscosímetro, obtenida por calibración (0,0928 s -1 ).t n = tiempo de escurrido (s).A partir de la tabla que se encuentra en anexo (Anexo 4), se lee el valor delproducto []×C, que corresponde al valor de la viscosidad relativa obtenido: rel = []×CCon:[] = viscosidad intrínseca (mL/g).C = concentración de la pasta seca en CED (g/mL).71

3. Material y MétodosDeterminación del contenido en ácidos hexenurónicosLa determinación del contenido de ácidos hexenurónicos (HexA) en las pastastratadas con POM se realizó según el procedimiento explicado anteriormente enel apartado 3.2.3.72

3. Material y Métodos73

4. ReferenciasREFERENCIASAbarca, R. y Blanco, M. L. (2008) Soda- Antraquinone Pulp of Tectona grandis inCosta Rica. V Congreso Iberoamericano de investigación en celulosa y papel,CIADICYP, Octubre 2008, Guadalajara, Jalisco, México.Adler, E. (1977) Lignin chemistry - Past, present and future. Wood Science andTechnology, 11, 169-218.Aitken, I., Cadel, F. y Voillot, C. (1988) Constituants fibreux des pates papiers etcartons pratique de l'analyse, 1st edition.Ali, M. y Sreekrishnan, T. R. (2001) Aquatic toxicity from pulp and paper milleffluents: A review. Advances in Environmental Research, 5, 175-196.Allen, L. H. (2000) Pitch control in paper mills. En: Pitch control, wood resin andderesination (Eds.: Back, E.L. y Allen, L.H.), TAPPI Press, Atlanta, pp. 307-328.Annergren G. E. (1996) Pulp bleaching. Principles and practice. Capítulo VII-3:Strength properties and characteristics of bleached chemical and(chemi)mechanical pulps. Ed. C. W. Dence y D. W. Reeve. Tappi press, pp:717-748Back, E. L. y Allen, L. H. (2000) Pitch control, wood resin and deresination.TAPPI Press, Atlanta.Bajpai, P., Anand, A., Sharma, N., Mishra, S. P., Pramod K. Bajpai P. K. yLachenal, D. (2006) Enzymes improve ECF Bleaching of pulp. BioResources, 1(1), 34-44.Bajpai, P. (1999) Application of enzymes in the pulp and paper industry.Biotechnology Progress, 15, 147-157. ISSN 8756-7938Bajpai, P. (2004) Biological bleaching of chemical pulps. Critical Reviews inBiotechnology, 24, 1-58.Bidlack, J., Malonge, M. y Benson, R. (1992) Molecular structure and componentintegration of secondary cell walls in plants. Proceedings of the Oklahoma.Academy of Science, 72, 51-56.Björkman, A. (1956) Studies on finely divided wood. Part I. Extraction of ligninwith neutral solvents. Svensk Papperstindning, 13, 477-485.Bertaud, F., Sundberg, A. y Holmbom, B. (2002). Evaluation of acid methanolysisfor analysis of wood hemicelluloses and pectins. Carbohydrate Polymers, 48,319-324.75

4. ReferenciasBland, D. E. (1966) Colorimetric and chemical identification of lignins in differentparts of Eucalyptus botryoides and their relation to lignification. Holzforschung,20, 12-16.Bleton, J., Mejanelle, P., Sansoulet, J., Goursaud, S. y Tchapla, A. (1996)Characterization of neutral sugars and uronic acids after methanolysis andtrimethylsilylation for recognition of plant gums. Journal of Chromatography A,720, 27-49.Boerjan, W., Ralph, J. y Baucher, M. (2003) Lignin biosynthesis. Annual Reviewof Plant Biology, 54, 519-546.Bourbonnais, R. y Paice, M. G. (1990) Oxidation of non-phenolic substrates. Anexpanded role for laccase in lignin biodegradation. FEBS Letters, 267, 99-102.Brett, C. y Waldron, K. (1996) Physiology and Biochemistry of Plant Cell Walls.Brooks, R. T., Edwards, L. L., Nepote, J. C. y Caldwell, M. R. (1994) Bleach plantcloseup and conversion to TCF: A case study using mill data and computersimulation. Tappi Journal, 77, 83-92.Brunow, G. (2001) Methods to reveal the structure of lignin. Biopolymers.Vol.1 -Lignin, Humic substances and Coal, 1, 89-116.Bryce J.R. (1990) Producción de pulpa al sulfito. En Pulpa y Papel: Química yTecnología Química. Vol. 1. Casey, J.P. Ed Limusa, México, 29-64.Call, H. P (1994) Verfahren zur Veränderung, Abbau oder Bleichen von Lignin,ligninhaltigen Materialien oder ähnlichen Stoffen. Patent (International) WO94/29510.Call, H. P. y Mücke, I. (1997) History, overview and applications of mediatedlignolytic systems, especially laccase-mediator-systems (Lignozym(R)-process).Journal of Biotechnology, 53, 163-202.Camarero, S., Sarkar, S., Ruiz-Dueñas, F. J., Martínez, M. J. y Martínez, A. T.(1999) Description of a versatile peroxidase envolved in natural degradation oflignin that has both Mn-peroxidase and lignin-peroxidase substrate binding sites.The Journal of Biological Chemistry, 274, 10324-10330Capanema, E. A., Balakshin, M. Y., Chen, C.-L., Gratzl, J. S. y Gracz, H. (2001)Structural analysis of residual and technical lignins by 1 H- 13 C correlation 2DNMR-spectroscopy. Holzforschung, 55, 302-308.Christierini, M., Ohlsson, A. B., Berglund, T. y Henriksson, G. (2005) Ligninisolated from primary walls of hybrid aspen cell cultures indicates significantdifferences in lignin structure between primary and secondary cell wall. PlantPhysiology and Biochemistry, 43, 777-785.76

4. Referenciasdel Río, J. C., Gutiérrez, A. y González-Vila, F. J. (1999) Analysis of impuritiesoccurring in a totally-chlorine free-bleached Kraft pulp. Journal ofChromatography A, 830, 227-232.del Río, J. C, Gutiérrez, A., González-Vila, F. J., Martín, F. y Romero, J. (1998)Characterization of organic deposits produced in the Kraft pulping of Eucalyptusglobulus wood. Journal of Chromatography A, 823,457-465del Río, J. C., Gutiérrez, A. y Martínez, A. T. (2004) Identifying acetylated ligninunits in non-wood fibers using pyrolysis-gas chromatography/massspectrometry. Rapid Communications in Mass Spectrometry, 18, 1181-1185.del Río, J. C., Marques, G., Rencoret, J., Martínez, A. T. y Gutiérrez, A. (2007)Occurrence of naturally acetylated lignin units. Journal of Agriculture and FoodChemistry, 55, 5461-5468.del Río, J. C., Rencoret, J., Marques, G., Gutiérrez, A., Ibarra, D., Santos, J. I.,Jiménez-Barbero, J., Zhang, L. y Martínez, A.T. (2008a) Highly acylated(acetylated and/or p-coumaroylated) native lignins from diverse herbaceousplants. Journal of Agriculture and Food Chemistry, 56, 9525-9534.del Río, J. C., Rencoret, J., Marques, G., Gutiérrez, A., Ibarra, D., Santos, J. I.,Jiménez-Barbero, J. y Martínez, A.T. (2008b) Occurrence and structuralcharacteristics of highly acylated (acetylated and/or p-coumaroylated) nativelignins from diverse herbaceous plants. 10 th European Workshop onlignocelluloses and Pulp. Stockholm Sweden, 26-29 August.del Río J. C., Romero J. y Gutiérrez A. (2000) Analysis of pitch deposits producedin Kraft pulp mills using a totally chlorine free bleaching sequence. Journal ofChromatography A, 874, 235-245Díaz, M. J., Alfaro, A., García, M. M., Eugenio, F. M. E., Ariza, J. y López, F.(2004) Ethanol pulping from tagasaste (Chamaecytisus proliferus L.F. ssp.Palmensis). A new promising source for cellulose pulp. Industrial &Engineering Chemistry Research, 43, 1875-1881.Evtuguin, D. V., Neto, C. P., Silva, A. M. S., Domingues, P. M., Amado, F. M. L.,Robert, D. y Faix, O. (2001) Comprehensive study on the chemical structure ofdioxane lignin from plantation Eucalyptus globulus wood. Journal ofAgricultural and Food Chemistry, 49, 4252-4261.Faix, O., Meier, D. y Fortmann, I. (1990) Thermal degradation products of wood.A collection of electron-impact (EI) mass spectra of monomeric lignin derivedproducts. Holz Roh-Werkstoff, 48, 351-354.FAO (2004) Base de datos de la FAO "FAOSTAT".77

4. ReferenciasFengel, D. (1989) Chemistry and morphology of quebracho colorado wood.International Symposium on Wood and Pulping Chemistry, Raleigh, 1-3.Fengel, D. y Wegener, G. (1984) Wood: Chemistry, ultrastructure, reactions. DeGruyter, Berlin.Fergus, B. J. y Goring, D. A. I. (1970) The location of guaiacyl and syringyllignins in birch xylem tissue. Holzforschung, 24, 113-117.Freudenberg, K. y Lehmann, B. (1963) Radioactive isotopes and lignin. X.Investigation of a lignin preparation labeled with 14 C. Chemische Berichte, 96,1850-1854.Freudenberg, K. y Neish, A. C. (1968) Constitution and biosynthesis of lignin.Springer-Verlag, New York.Fuji, K., Ichikawa, K., Node, M. y Fujita, E. (1979) Hard acid and soft nucleophilesystem. New efficient method for removal of benzyl protecting group. Journalof Organic Chemistry, 44, 1661-1664.Fujita, Y., Awaji, H., Matsukura, M. y Hata, K. (1991) Enzymic pitch control inpapermaking process. Kami Pa Gikyoshi, 45, 905-921Fujita, Y., Awaji, H., Taneda, H., Matsukura, M., Hata, K., Shimoto, H., Sharyo,M., Sakaguchi, H. y Gibson, K. (1992) Recent advances in enzymic pitchcontrol. Tappi Journal, 75 (4), 117-122Fukushima, K. y Terashima, N. (1991) Heterogeneity in formation of lignin. XIV.Formation and structure of lignin in differentiating xylem of Ginkgo biloba.Holzforschung, 45, 87-94.Fullerton, T. J. y Franich, R. A. (1983) Lignin analysis by pyrolysis-GC-MS.Holzforschung, 37, 267-269.Gamelas J. A. F., Cavaleiro A., Santos I., Balula M. 2003. Os polioxometalatos.Do anião de Keggin às nanocápsulas. Boletim da Sociedade Portuguesa deQuímica. 90, 45-51.Gamelas, J. A. F., Evtuguin, D. V. y Gaspar, A. R. (2008) Transition metalcomplexes in the delignification catalysis. In: Varga, B., Kis, L. (Eds.),Transition metal chemistry: New research. Nova Science Publishers, Inc., NewYork, pp. 15-57.Gamelas J. A. F., Pontes A. S. N., Evtuguin D. V., Xavier A. M. R. B. y EsculcasA. P. (2007) New polyoxometalete-laccase integrated sistem for kraft pulpdelignification. Biochemical Engineering Journal, 33, 141-147.78

4. ReferenciasGaspar, A. R., Gamelas, J. A. F., Evtuguin, D. V. y Neto, C. P. (2007) Alternativesfor lignocellulosic pulp delignification using polyoxometalates and oxygen: areview. Green Chemistry, 9, 717-730.García Hortal. (2007) Fibras Papeleras. Ediciones UPC (Universitat Politècnica deCatalunya), Terrassa (Spain).García Hortal, J. A. y Colom, J. F. (1992) El proceso al sulfato. Vol. I. UniversitatPolitècnica de Catalunya, Terrassa (Spain).García, M. M., López, F., Alfaro, A., Ariza, J., Tapias, R. (2008) The use oftagasaste (Chamaecytisus proliferus) from different origins for biomass andpaper production. Bioresource technology, 99(9), 3451-7.Gellerstedt, G. y Li, J. (1996) An HPLC method for the quantitative determinationof hexenuronic acid groups in chemical pulps. Carbohydrate Research, 294, 41-51.Gellerstedt, G. Pranda J. y Lindfors E-L. (1994) Structural and molecularproperties of residual birch kraft lignins. Journal of Wood Chemistry andTechnology, 14(4), 467-482.Gilarranz, M. A., Oliet, M., Rodríguez, F. and Tijero, J. (1999) Methanol-basedpulping of Eucalyptus globulus. Canadian Journal of Chemical Engineering, 77,515-521.Glenn, J. K., Morgan, M. A., Mayfield, M. B., Kuwahara, M. y Gold, M. H. (1983)An extracellular H 2 O 2 -requiring enzyme preparation involved in ligninbiodegradation by the write rot basidiomycete Phanerochaete chrysosporium.Biochemical and Biophysical Research Communications, 114, 1077-1083.Grabber, J. H., Quideau, S. y Ralph, J. (1996) p-Coumaroylated syringyl units inmaize lignin: Implications for -ether cleavage by thioacidolysis.Phytochemistry, 43, 1189-1194.Gutiérrez, A. y del Río, J. C. (2001) Gas chromatography-mass spectrometrydemonstration of steryl glycosides in eucalypt wood, kraft pulp and processliquids. Rapid Communications in Mass Spectrometry, 15, 2515-2520.Gutiérrez, A. y del Río, J. C. (2005) Pitch episodes produced during themanufacturing of high-quality paper pulps from hemp fibers. BioresourseTechnology, 96, 1445-1450.Gutiérrez, A., del Río, J. C., González-Vila, F. J. y Martín, F. (1998) Analysis oflipophilic extractives from wood and pitch deposits by solid-phase extractionand gas chromatography. Journal of Chromatography A, 823, 449-455.79

4. ReferenciasGutiérrez, A., del Río, J. C., González-Vila, F. J. y Martín, F. (1999) Chemicalcomposition of lipophilic extractives from Eucalyptus globulus Labill. wood.Holzforschung, 53, 481-486.Gutiérrez, A., del Río, J. C., Ibarra, D., Rencoret, J., Romero, J., Speranza, M.,Camarero, S., Martínez, M. J. y Martínez, A. T. (2006a) Enzymatic removal offree and conjugated sterols forming pitch deposits in environmentally soundbleaching of eucalypt paper pulp. Environmental Science and Technology, 40,3416-3422.Gutiérrez, A., del Río, J. C. y Martínez, A. T. (2004) Chemical analysis andbiological removal of wood lipids forming pitch deposits in paper pulpmanufacturing. In Spencer, J. F. T. (ed), Protocols in EnvironmentalMicrobiology. Humana Press, Totowa, USA.Gutiérrez, A., del Río, J. C. y Martínez, A. T. (2009) Microbial and enzymaticcontrol of pitch in the pulp and paper industry. Applied Microbiology andBiotechnology, 82, 1005-1018.Gutiérrez, A., del Río, J. C., Martínez, M. J. y Martínez, A. T. (2001) Thebiotechnological control of pitch in paper pulp manufacturing. Trends inBiotechnology, 19, 340-348.Gutiérrez, A., del Río, J. C., Rencoret, J., Ibarra, D. y Martínez, A. T. (2006b)Main lipophilic extractives in different paper pulp types can be removed usingthe laccase-mediator system. Applied Microbiology and Biotechnology, 72, 845-851.Gutiérrez, A., del Río, J. C., Rencoret, J., Ibarra, D., Speranza, A. M., Camarero,S., Martínez, M. J. y Martínez, A. T. (2006c) Sistema enzima-mediador para elcontrol de los depósitos de pitch en la fabricación de pasta y papel. Patent(International) ES2282020B1.Gutiérrez, A., Rencoret, J., Ibarra, D., Molina, S., Camarero, S., Romero, J., delRío, J. C. y Martínez, A. T. (2007) Removal of lipophilic extractives from paperpulp by laccase and lignin-derived phenols as natural mediators. EnvironmentalScience & Technology, 41, 4124-4129.Hamberg, M., Su, C. y Oliw, E. (1998) Manganese lipoxygenase - Discovery of abis-allylic hydroperoxide as product and intermediate in a lipoxygenase reaction.The Journal of Biological Chemistry, 273, 13080-13088.Han, J. S. (1998) Properties of Nonwood Fibers. Proceedings of the KoreanSociety of Wood Science and Technology Annual Meeting.80

4. ReferenciasHardell, H. L., Leary, G. J., Stoll, M. y Westermark, U. (1980a) Variations inlignin structure in defined morphological parts of birch. Svensk Papperstidn, 83,71-74.Hardell, H. L., Leary, G. J., Stoll, M. y Westermark, U. (1980b) Variations inlignin structure in defined morphological parts of spruce. Svensk Papperstidn,83, 44-49.Hata, K., Matsukura, M., Taneda, H. y Fujita, Y. (1996) Mill-scale application ofenzymatic pitch control during paper production. In: Viikari, L. and Jeffries, T.W. (eds) Enzymes for pulp and paper processing. ACS, Washington, pp 280-296.Higuchi, T. (1997) Biochemistry and molecular biology of wood. Springer Verlag,London.Hillis, W. E. (1962) Wood extractives. Academic Press, London.Hillis, W. E. y 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.Hoareau, W., Trindade, W. G., Siegmund B., Castellan, A. y Frollini E. (2004)Sugar cane bagasse and curaua lignins oxidized by chlorine dioxide and reactedwith furfuryl alcohol: characterization and stability. Polymer Degradation andStability, 86, 567-576.Irie, Y. (1990) Enzymatic pitch control in papermaking system. Abs 1990Papermakers Conference, 23-25 April, Atlanta.Jiménez, L., Pérez, A., Rodríguez, A. y de La Torre, M.J. (2006) New rawmaterials and pulping processes for production of pulp and paper. Afinidad 63(525), 362-369.Jiménez, L., Pérez, A., de la Torre, M.J., Moral A. y Serrano, L. (2007)Characterization of vine shoots, cotton stalks, Leucaena leucocephala andChamaecytisus proliferus, and of their ethyleneglycol pulps. BioresourceTechnology, 98, 3487-3490.Jones, J. B. y Case, V. W. (1990) Sampling, handling, and analyzing plant tissuesamples. Soil Testing and Plant Analysis, 389-427.Karhunen, P., Rummakko, P., Sipila, J., Brunow, G. y Kilpeläinen, I. (1995)Dibenzodioxocins - A novel type of linkage in softwood lignins. TetrahedronLetters, 36, 169-170.81

4. ReferenciasKawai, S., Umezawa, T. y Higuchi, T. (1987a) p-Benzoquinone monoketals, noveldegradation products of -O-4 lignin model compounds by Coriolus versicolorand lignin peroxidase of Phanerochaete chrysosporium. FEBS Letters, 210, 61-65.Kawai, S., Umezawa, T., Shimada, M., Higuchi, T., Koide, K., Nishida, T.,Morohoshi, N. y Haraguchi, T. (1987b) C -C cleavage of phenolic -1 ligninsubstructure model compound by laccase of Coriolus versicolor. MokuzaiGakkaishi, 33, 792-797.Kuwahara, M., Glenn, J. K., Morgan, M. A. Y Gold, M. H. (1984) Separation andcharacterization of two cellular H 2 O 2 -dependent oxidases from ligninolyticcultures of Phanerochaete chrysosporium. FEBS Letters, 1969, 247-250.Lapierre, C., Monties, B. y Rolando, C. (1985) Thioacidolysis of lignin:Comparison with acidolysis. Journal of Wood Chemistry and Technology, 5,277-292.Lapierre, C., Monties, B. y Rolando, C. (1986) Preparative thioacidolysis of sprucelignin: Isolation and identification of main monomeric products. Holzforschung,40, 47-50.Lapierre, C., Pollet, B. y Monties, B. (1991) Thioacidolysis of spruce lignin: GC-MS analysis of the main dimers recovered after Raney nickel desulphuration.Holzforschung, 45, 61-68.Leao, A. L., Rowell, R. y Tavares, N. (1998) Applications of natural fibres inautomotive industry in Brazil-Thermoforming process. In: Science andTechnology of Polymers Advanced Materials. Emerging Technologies andBusiness Opportunities; Prasad, P. N., Mark, J. E., Kandil, S. H., Kafafi, Z. H.,Eds., Plenum Press: New York, 755-761.Lennholm, H. y Henriksson, G. (2007) Chapter 4. Cellulose. In Fibre and PolymerTechnology, K. I. I. (ed), Ljungberg Textbook Pulp and Paper Chemistry andTechnology Book 1. Wood Chemistry and Technology.Liitiä, T. M., Maunu, S. L., Hortling, B., Toikka, M. y Kilpeläinen, I. (2003)Analysis of technical lignins by two- and three-dimensional NMR spectroscopy.Journal of Agricultural and Food Chemistry, 51, 2136-2143.Lin, S. Y. y Dence, C. W. (1992) Methods in lignin chemistry. Springer-Verlag,Berlin.López, F., Alfaro, A., García, M.M., Diaz, M.J., Calero, A.M. y Ariza, J. (2004)Pulp and paper from tagasaste (Chamaecytisus proliferus ssp palmensis).Chemical Engineering Research and Design, 82, 1029-1036.82

4. ReferenciasLu, F. y Ralph, J. (1997a) Derivatization followed by reductive cleavage (DFRCmethod), a new method for lignin analysis: protocol for analysis of DFRCmonomers. Journal of Agricultural and Food Chemistry, 45, 2590-2592.Lu, F. y Ralph, J. (1997b) The DFRC method for lignin analysis. Part 1. A newmethod for aryl ether cleavage: lignin model studies. Journal of Agriculturaland Food Chemistry, 45, 4655-4660.Lu, F. y Ralph, J. (1998) The DFRC method for lignin analysis. 2. Monomers fromisolated lignin. Journal of Agricultural and Food Chemistry, 46, 547-552.Martínez A. T., Rencoret J., Marques G., Gutiérrez A., Ibarra D., Jiménez-BarberoJ., and del Río J. C. (2008) Monolignol acylation and lignin structure in somenonwoody plants: A 2D-NMR study. Phytochemistry, 69, 2831-2843.Martínez, M. J., Ruiz-Dueñas, F. J., Guillén, F. y Martínez, A. T. (1996)Purification and catalytic properties of two manganese-peroxidase isoenzymesfrom Pleurotus eryngii. European Journal of Biochemistry, 237, 424-432.Matsukura, M., Fujita, Y. y Sakaguchi, H. (1990) On the use of Resinase forpitch control. Novo Publication. A, 6122, 1-7.Mayer, A. M. y Staples, R. C. (2002) Laccase: new functions for an old enzyme.Phytochemistry, 60, 551-565.McDougall, G. J., Morrison, I. M., Stewart, D., Weyers, J. D. B. y Hillman, J. R.(1993) Plant fibres: Botany, chemistry and processing for industrial use. Journalof the Science of Food and Agriculture, 62, 1-20.Meier, D. y Faix, O. (1992) Pyrolysis-gas chromatography-mass spectrometry. InLin, S. Y. and Dence, C. W. (eds), Methods in lignin chemistry. Springer-Verlag, Berlin, pp. 177-199.Molina, S., Rencoret, J., del Río, J. C., Lomascolo, A., Record, E., Martínez, A. T.y Gutiérrez, A. (2008) Oxidative degradation of model lipids representative formain paper pulp lipophilic extractives by the laccase-mediator system. AppliedMicrobiology and Biotechnology, 80, 211-222.Morrison, W. H. III., Akin, D. E., Himmelsbach, D. S. y Gamble, G. R. 1999.Chemical, microscopic, and instrumental analysis of graded flax fibre and yarn.Journal of the Science of Food and Agriculture, 79, 3-10.Neto, C. P., Seca, A. M. L., Fradinho, D., Coimbra, M. A., Domingues, F. M. J.,Evtuguin, D., Silvestre, A. J. D. y Cavaleiro, J. A. S. (1996) Chemicalcomposition and structural features of the macromolecular components ofHibiscus cannabinus grown in Portugal. Industrial Crops and Products, 5, 189-196.83

4. ReferenciasNode, M., Hori, H. y Fujita, E. (1976) Demethylation of aliphatic methyl etherswith a thiol and boron trifluoride. Journal of the Chemical Society, PerkinTransactions, 1, 2237-2240.Norma Finlandesa TYÖOHJE C/6 15/07/98.Paice, M. G., Bourbonnais, R., Reid, I. D., Archibald, F. S. y Jurasek, L. (1995)Oxidative bleaching enzymes: A review. Journal of Pulp and Paper Science, 21,J280-J284.Pande, H. y Roy, D. N (1998) Influence of fibre morphology and chemicalcomposition on the papermaking potential of kenaf fibres. Pulp & PaperCanada, 99(11), 31-34.Parhan A., Gray R. L. (1990) The practical identification of wood pulp fibers.Tappi Press. USA.Pérez-Boada, M., Ruiz-Dueñas, F. J., Pogni, R., Basosi, R., Choinowski, T.,Martínez, M. J., Piontek, K. y Martínez, A. T. (2005) Versatile peroxidiseoxidation of high redox potential aromatic compounds: site-directedmutagenesis, spectroscopic and crystallographic investigations of three longrangeelectrón transfer pathways. Journal of Molecular Biology, 354, 385-402.Pope M. T. Springer Verlag. (1983) Heteropoly and Isopoly Oxometalates.Inorganic Chemistry Concepts, V 8. Berlin.Ralph, J. y Hatfield, R. D. (1991) Pyrolysis-GC-MS characterization of foragematerials. Journal of Agricultural and Food Chemistry, 39, 1426-1437.Ralph, J., Hatfield, R. D., Piquemal, J., Yahiaoui, N., Pean, M., Lapierre, C. yBoudet, A. M. (1998) NMR characterization of altered lignins extracted fromtobacco plants down-regulated for lignification enzymes cinnamyl-alcoholdehydrogenase and cinnamoyl-CoA reductase. Proceedings of the NationalAcademy of Sciences, 95, 12803-12808.Ralph, J., Lapierre, C., Lu, F. C., Marita, J. M., Pilate, G., van Doorsselaere, J.,Boerjan, W. y Jouanin, L. (2001) NMR evidence for benzodioxane structuresresulting from incorporation of 5-hydroxyconiferyl alcohol into lignins of O-methyltransferase-deficient poplars. Journal of Agricultural and FoodChemistry, 49, 86-91.Ralph, J. y Lu, F. (1998) The DFRC method for lignin analysis. 6. A simplemodification for identifying natural acetates in lignin. Journal of Agriculturaland Food Chemistry, 46, 4616-4619.84

4. ReferenciasRalph, J., MacKay, J. J., Hatfield, R. D., Omalley, D. M., Whetten, R. W. ySederoff, R. R. (1997) Abnormal lignin in a loblolly pine mutant. Science, 277,235-239.Rigol, A., Latorre, A., Lacorte, S. y Barceló, D. (2004) Bioluminescence inhibitionassays for toxicity screening of wood extractives and biocides in paper millprocess waters. Environmental Toxicology and Chemistry, 23, 339-347.Robert, D. (1992) Carbon-13 nuclear magnetic resonance. In Lin, S. Y. and Dence,C. W. (eds), Methods in lignin chemistry. Springer-Verlag, Berlin, pp. 250-273.Rolando, C., Monties, B. y Lapierre, C. (1992) Thioacidolysis. In Lin, S. Y. andDence, C. W. (eds), Methods in lignin chemistry. Springer-Verlag, Berlin, pp.334-349.Rowe, J. W. (1989) Natural products of woody plants. I and II. Chemicalsextraneous to the lignocellulosic cell wall. Springer-Verlag, Berlin.Ruiz-Dueñas, F. J. y Martínez, A. T. (2010) Structural and functional features ofperoxidases with a potential as industrial biocatalysts. In: Torres, E. y Ayala, M.(eds.). Biocatalysis Based on Heme Peroxidases (ISBN: 978-3-642-12626-0).Springer.Ruiz-Dueñas, F. J., Martínez, M. J. y Martínez, A. T. (1999a) Heterologousexpression of Pleurotus eryngii peroxidase confirms its ability to oxidize Mn 2+and different aromatic substrates. Applied and Environmental Microbiology, 65,4705-4707.Ruiz-Dueñas, F. J., Martínez, M. J. y Martínez, A. T. (1999b) Molecularcharacterization of a novel peroxidase isolated from the ligninolytic fungusPleurotus eryngii. Molecular Microbiology, 31, 223-236.Santos, A., Rodríguez, F., Gilarranz, M. A., Moreno, D. y García-Ochoa, F. (1997)Kinetic modeling of kraft delignification of Eucalyptus globulus. Industrial &Engineering Chemistry Research, 36, 4114-4125.Saito, K. y Fukushima, K. (2005) Distribution of lignin interunit bonds in thedifferentiating xylem of compression and normal woods of Pinus thunbergii.Journal of Wood Science, 51, 246-251.Sarkanen, K. V. y Hergert, H. L. (1971) Classification and distribution. InSarkanen, K. V. and Ludwig, C. H. (eds), Lignins - Occurrence, Formation,Structure and Reactions. Wiley-Interscience, New York, pp. 43-94.Scandinavian Pulp Paper and Board Committee, 1994. SCAN Test Methods.Sweden.85

4. ReferenciasSeca, A. M., Cavaleiro, J. A. S., Domingues, F. M. J., Silvestre, A. J. D, Evtuguin,D. y Neto, C. P. (2000) Structural characterization of the lignin from the nodesand Internodes of Arundo donax Reed. Journal of Agricultural and FoodChemistry, 48, 817-824.Sederoff, R. R., MacKay, J. J., Ralph, J. y Hatfield, R. D. (1999) Unexpectedvariation in lignin. Current Opinion in Plant Biology, 2, 145-152.Selvendran, R. R., March, J. F. y Ring, S. G., (1979). Determination of aldoses anduronic acids content of vegetable fiber. Analytical Biochemistry, 96, 282-292.Shatalov, A. A. y Pereira, H. (2002) Influence of stem morphology on pulp andpaper properties of Arundo donax L. reed. Industrial Crops and Products, 15,77-83.Shimizu, K. (2001) Wood and Cellulosic Chemistry, 2nd edition, Eds. D.N.-S.Hon, N. Shiraishi, Marcel Dekker Inc., New York, USA, 177-214.Sigoillot, C., Camarero, S., Vidal, T., Record, E., Asther, M., Pérez-Boada, M.,Martínez, M.J., Sigoillot, J.C., Asther, M., Colom, J. y Martínez, A.T. (2005)Comparison of different fungal enzymes for bleaching high-quality paper pulps.Journal of Biotechnology, 115, 333–34.Silva, G. S., Assis, M. B. y Barbosa, W. L. R. (2001) Investigaçao fitoquímica emicrobiologica da espécie Ananas erectifolius (curauá). ReV. Virtual IniciaçaoAcad. UFPA, 1.Sjöström, E. y Westermark, U. (1999) Chemical Composition of Wood and Pulps:Basic constituents and Their Distribution. In: Analytical Methods in WoodChemistry, Pulping and Papermaking. E. Sjöström and R. Alén (Eds.), Springer-Verlag, Berlin, Germany, Chap. 1, pp. 1-19.Sjöström, E. (1981) Wood Chemistry: Fundamentals and Applications. AcademicPress, Orlando, pp. 68–82.Sjöström, E. (1993) Wood chemistry. Fundamentals and Applications. AcademicPress, San Diego.Streitwieser, A. Jr. y Heathcock, C. H. (1983). Química Orgánica. 3ª edición.Importecnica, S.A. 726-730.Struik, P. C., Amaducci, S., Bullard, M. J., Stutterheim, N. C., Venturi G. yCromack, H. T. H. (2000) Agronomy of fibre hemp (Cannabis sativa L.) inEurope. Industrial Crops and Products, 11, 107-118.Su, C. y Oliw, E. (1998) Manganese lipoxygenase - Purification andcharacterization. The Journal of Biological Chemistry, 273, 13072-13079.86

4. ReferenciasSundberg, A., Sundberg, K., Lillandt, C. y Holmbom, B. (1996). Determination ofhemicelluloses and pectins in wood and pulp fibers by acid methanolysis andgas chromatography. Nordic Pulp and Paper Research Journal, 4, 216-226.Tappi (2004) 2004-2005 TAPPI Test Methods. TAPPI Press, Norcoss, GA 30092,USA.Tappi (2006) 2006-2007 TAPPI Test Methods. TAPPI Press, Norcoss, GA 30092,USA.Tavares, A. P. M., Gamelas, J. A. F., Gaspar, A. R., Evtuguin, D. V. y Xavier, A.M. R. B. (2004) A novel approach for the oxidative catalysis employingpolyoxometalate-laccase system: application to the oxygen bleaching of kraftpulp. Catalysis Communications, 5, 485-489.Tien, M. y Kirk, T. K. (1983) Lignin-degrading enzyme from the hymenomycetePhanerochaete chrysosporium Burds. Science, 221, 661-663.Viikari, L., Kantelinen, A., Sundquist, J. y Linko, M. (1994) Xylanases inbleaching - From an idea to the industry. FEMS Microbiology Reviews, 13, 335-350.Vuorinen, T., Fagerström, P., Buchert, J., Tenkanen, M., y Teleman, A. (1999).Selective hydrolysis of hexenuronic acid groups and its application in ECF andTCF bleaching in kraft pulps. Journal of Pulp and Paper Science, 25(5), 155-162.Watanabe, T. (2003) Analysis of native bonds between lignin and carbohydrate byspecific chemical reactions. In Springer-Verlag, B. H. (ed), Timell, T.E. (Ed.).Springer series in wood science, Association between lignin and carbohydratesin wood and other plant tissues. pp. 91-130.Weinstock, I. A., Atalla, R.H., Reiner, R. S., Moen, M.A. Hammel, K. E.,Houtman, C. J., Hill, C. L. y Harrup, M. K. (1997) A new environmentallybening technology for transforming wood pulp into paper. Engineeringpolyoxometalates as catalysts for multiple processes. Journal of MolecularCatalysis A: Chemical, 116, 59-84.Zhang, L. y Gellerstedt, G. (2001) NMR observation of a new lignin structure, aspiro-dienone. Chemical Communications, 2744-2745.Zhang, L. M. y Gellerstedt, G. (2007) Quantitative 2D HSQC NMR determinationof polymer structures by selecting suitable internal standard references.Magnetic Resonance in Chemistry, 45, 37-45.87

4. ReferenciasZhang, L. M., Gellerstedt, G., Ralph, J. y Lu, F. C. (2006) NMR studies on theoccurrence of spirodienone structures in lignins. Journal of Wood Chemistry andTechnology, 26, 65-79.Zhang, X., Nguyen, D., Paice, M. G., Tsang, A. y Renaud, S. (2007) Degradationof wood extractives in thermo-mechanical pulp by soybean lipoxygenase.Enzyme and Microbial Technology, 40, 866-873.88

4. Referencias89

5. Resultados y discusiónRESULTADOS Y DISCUSIÓNLos resultados obtenidos durante la Tesis y la discusión de los mismos semuestran a continuación en forma de publicaciones. Durante la realización deesta Tesis se han publicado los resultados principales de los estudios realizadossobre: i) la composición química de la lignina, lípidos y hemicelulosas dediversos cultivos lignocelulósicos (Publicaciones I-VI); ii) el comportamiento delos constituyentes orgánicos de diversos cultivos lignocelulósicos durante lafabricación de pastas de papel de alta calidad, que incluyen procesos de cocciónsosa-AQ y blanqueo TCF y ECF (Publicaciones VII-VIII); y iii) el desarrollo dedos procedimientos biotecnológicos para la degradación de lignina y lípidosresiduales en pastas de papel (Publicaciones IX-X).91

5. Resultados y discusiónPublicación I:Marques G., Rencoret J., Gutiérrez A., del Río J.C. (2010) Evaluation of thechemical composition of different non-woody plant fibers used for pulp and papermanufacturing. The Open Agriculture Journal (in press).92

5. Resultados y discusiónEvaluation of the chemical composition of different non-woody plant fibersused for pulp and paper manufacturingGisela Marques, Jorge Rencoret, Ana Gutiérrez, José C. del RíoInstituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, P.O. Box 1052, 41080-Seville, SpainAbstractThe chemical composition of several non-woody plant fibers (bast fibers fromflax, 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 andthe structure of the lignin polymer since they are important components of thefiber that strongly influence the pulping and bleaching performances.Keywords: non-woody fibers; flax, hemp, kenaf, jute, sisal, abaca, giant reed,paper pulp; lipophilic extractives; lignin1. IntroductionAn alternative to woody raw materials for pulp and paper production indeveloping countries is the use of non-woody fibers from field crops andagricultural residues. In developed countries, non-woody fibers are mainly usedfor 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 consideralternative agricultural strategies that move an agricultural industry purelyfocused on food production to one that also supplies the needs of other industrialsectors, such as paper and textiles. Non-wood fibers, therefore, could becomeimportant raw materials in this transformation [1-3]. The main sources of nonwoodyraw materials are agricultural residues from monocotyledons, includingcereal straw and bagasse, or plants grown specifically for the fiber, such asbamboo, reeds, and some other grass plants such as flax, hemp, kenaf, jute, sisal,or abaca. Non-woody plants offer several advantages including short growthcycles, moderate irrigation requirements and low lignin content, which inprinciple would result in reduced energy and chemicals consumption duringpulping [4].Plant fibers are constituted by three structural polymers (the polysaccharidescellulose, and hemicelluloses and the aromatic polymer lignin) as well as bysome minor non-structural components (i.e. proteins, extractives, minerals).Pulping and bleaching performances are highly dependent on the relativecontent, structure and reactivity of the plant components. In particular, the lignincontent and its composition in terms of p-hydroxyphenyl (H), guaiacyl (G) andsyringyl (S) moieties and the different inter-unit linkages are important factors93

5. Resultados y discusiónin pulp production affecting the delignification rate. It has been shown thathigher S/G ratios in woods implied higher delignification rates, less alkaliconsumption and therefore higher pulp yield [5]. On the other hand, among thenon-structural components, lipophilic extractives present special relevance dueto their high impact in paper pulp manufacturing [6]. Lipophilic extractivesinclude different classes of compounds (i.e. alkanes, fatty alcohols, fatty acids,free and conjugated sterols, terpenoids, triglycerides and waxes), which havedifferent behavior during pulping and bleaching [6-8]. These lipophiliccompounds, even when present in low amounts in the raw material, may play animportant role during the industrial wood processing for pulp and paperproduction since they are at the origin of the so-called pitch deposits. Pitchdeposition is a serious problem in the pulp and paper industry being responsiblefor reduced production levels, higher equipment maintenance costs, higheroperating 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 paperpulp production, a more complete understanding of its chemistry is required.Most studies have been devoted to the chemical characterization of woodymaterials, while studies on non-woody fibers have been comparatively scarce. Inthis context, the main objective of this work is to revise and evaluate thechemical composition of different non-woody plant fibers used for pulp andpapermaking, that will help improving the industrial processes in which they areused as raw materials.2. Analytical methodologies2.1. SamplesThe samples selected for this study were bast fibers from flax (Linumusitatissimum), hemp (Cannabis sativa), kenaf (Hibiscus cannabinus) and jute(Corchorus capsularis); leaf fibers from sisal (Agave sisalana), abaca (Musatextilis) and curaua (Ananas erectifolius); as well as giant reed (Arundo donax).2.2. Chemical analysesFor hemicellulose and Klason lignin content estimation, milled samples wereextracted with acetone in a Soxhlet apparatus for 8h and subsequently extractedwith hot water (3h at 100 ºC). The acetone extracts were evaporated to drynessand resuspended in chloroform for chromatographic analysis of the lipophilicfraction. Klason lignin was estimated as the residue after sulfuric acid hydrolysisof the pre-extracted material. The acid-soluble lignin was determined, afterfiltering off the insoluble lignin, by spectrophotometric determination at 205 nmwavelength. Neutral sugars from polysaccharide hydrolysis were analyzed asalditol acetates by GC according to Tappi rules T222 om-88 and T249 om85,respectively [9]. Ash content was estimated as the residue after 6h at 575 C.94

5. Resultados y discusión2.3. Analysis of lipidsThe broad range of molecular masses of lipophilic extractives and theirstructural diversity represent two important difficulties for their chemicalanalysis. High-temperature, short-length (5m) capillary columns with thin filmswas used for the rapid identification and quantification of lipophilic woodextractives with no prior derivatization nor fractionation [10], resulting in anoptimal analysis of high-molecular-weight lipids such as waxes, sterol esters,and triglycerides. This method enables elution and separation of compoundswith a wide range of molecular weights (from fatty and resin acids to sterolesters and triglycerides) in the same chromatographic analysis. For GC-MS,medium-length high-temperature capillary columns (12 m) were used [10].When a more accurate characterization of some compounds was required, theextracts were fractionated by solid-phase extraction (SPE) procedures [10-11].2.4. Analysis of ligninPyrolysis coupled to gas chromatography/mass spectrometry (Py-GC/MS) wasused for the “in situ” analysis of the chemical composition of the lignin, in termsof their H:G:S distribution. Lignin is thermally degraded to produce a mixture ofrelatively simple phenols, which result from cleavage of ether and certain C-Cinter-unit linkages. The released methoxylated phenols retain the substitutionpatterns of the different lignin monomers, and it is thus possible to identifycomponents from the p-hydroxyphenylpropanoid (H), guaiacylpropanoid (G)and syringylpropanoid (S) lignin units [5, 12, 13]. For a more detailed structuralstudy, the milled wood lignins were isolated according to a known procedure[14] and analyzed by bidimensional nuclear magnetic resonance (2D-NMR).2D-NMR can provide information of the structure of the whole macromoleculeand is a powerful tool for lignin structural elucidation since signals overlappingin the 1 H and 13 C NMR spectra are resolved revealing both the aromatic unitsand the different interunit linkages present in lignin [15-18].3. Characterization of the selected non-woody plant fibers3.1. Morphological characteristics of the fibersThe morphological characteristics of a fiber, such as fiber length and width, areimportant parameters in estimating pulp qualities. Fiber length is the mostimportant physical property for pulping as it generally influences the tearingstrength of paper. Greater the fiber length, higher will be the tearing resistanceof paper. On the other hand, longer fibers tend to give a more open and lessuniform sheet structure. Table 1 shows the morphological characteristics of thenon-woody fibers used for this study [19]. An important feature of non-woodfibers is the wide variability among the lengths of the fibers of different species.Some of these fibers have short lengths (i.e. giant reed, with only 1180 m fiber95

5. Resultados y discusiónlength), similar to the short fibers of hardwoods, while others, and particularlyflax and hemp bast fibers, present remarkably high lengths (up to 28000 mfiber length).Table 1. Morphological characteristics (length and width) of the selected fibers [19].Fiber Source Length (m) Width (m) L/W ratioBast fibersflax 28000 21 1350:1hemp 20000 22 1000:1kenaf 2740 20 135:1jute 2000 20 100:1Leaf fiberssisal 3030 17 180:1abaca 6000 20 300:1curaua n.a. n.a. n.a.Reedsgiant reed 1180 15 78:1Woods for comparisonsoftwoods 3000 30 100:1hardwoods 1250 25 50:1n.a. not availableAmong the studied fibers, flax and hemp pulps have traditionally been used asthe primary furnish for cigarette paper (burning tube), where strength, opacityand control of air permeability are required. Banknote paper often incorporatesflax or hemp to enhance general strength characteristics. Jute pulp is used forhigh porosity papers. Its fiber length plus low diameter makes it very suitable forfinishing paper purposes. Sisal and abaca pulps have an unusually high tearingresistance and high porosity and are well suited for the production of paperswhere high strength and high porosity are required.3.2. Raw chemical composition of the fibersThe chemical composition of the main constituents of the selected non-woodyfibers is shown in Table 2. In general, they are characterized by a highpolysaccharide content and low contents of lignin, lipids and ash [20-22]. Giantreed presents the lowest holocellulose content and the higher content of lignin,which makes it less interesting for pulp and papermaking. The low lignincontent of the rest of the fibers, with a lignin content as low as 2.9 % in flax, isin principle advantageous for pulping. Moreover, the acetone extractives contentis also low, and usually less than 2%, except for curaua fibers (5.3% of total96

5. Resultados y discusiónfiber weight). However, most of the acetone extracts in curaua corresponds topolar compounds, while only 1.3% corresponds to lipophilic compounds, whichwere estimated by redissolving the acetone extracts in chloroform. Thus, ingeneral, the lipophilic content of the selected non-woody fibers ranges from 0.5to 1.3%. Finally, the ash content for all the selected fibers was low incomparison to other raw materials used for pulp and papermaking, as the cerealstraws, with an ash content generally higher than 15% [1]. Therefore, accordingto their chemical composition, most of these fibers seem suitable raw materialsfor pulp and papermaking.Table 2. Composition of the main constituents of the selected fibers (% of dry matter) [20-22].Ash AcetoneextractivesWater-solubles KlasonligninAcid-solubleligninHolocelluloseBast fibersflax 1.5 0.7 1.3 2.9 1.6 92.0hemp 2.0 0.5 1.2 4.6 1.5 90.3kenaf 1.8 1.0 1.1 11.4 3.0 81.9jute 2.4 0.5 0.4 13.3 2.8 81.6Leaf fiberssisal 1.0 0.7 2.3 5.9 3.0 85.0abaca 0.9 0.5 1.7 7.7 1.4 85.6curaua 1.3 5.3 5.1 4.9 1.6 92.5Reedsgiant reed 4.2 1.6 8.5 24.7 n.d. 49.83.3. Carbohydrate composition of the fibersThe results of the analyses of neutral sugars of the non-woody fibers selected forthis study are reflected in Table 3. The hemicelluloses fraction of the bast fiberspresents a higher variability than those of the leaf fibers. Thus, hemicellulosesfrom flax and hemp are mainly constituted by mannose followed by galactose,while the hemicelluloses from kenaf and jute are predominantly constituted byxylose. On the other hand, all the leaf fibers (sisal, abaca and curaua) show apredominance of xylose. Finally, giant reed presents a strikingly high content ofxylose, that amounts up to 39.2% of the total neutral sugars.97

5. Resultados y discusiónTable 3. Composition of neutral monosaccharides (as percentage of total neutralcarbohydrates) [20-22].Rhamnose Arabinose Xylose Mannose Galactose GlucoseBast fibersflax 0.4 0.9 1.1 8.8 3.5 85.3hemp 0.4 0.6 1.0 9.9 1.6 86.4kenaf 0.5 2.1 10.5 4.9 0.5 81.5jute 0.5 1.5 7.9 4.2 0.5 85.4Leaf fiberssisal 0.3 1.9 12.0 3.6 0.6 81.7abaca 0.3 1.6 7.5 3.5 0.3 86.9curaua 0.0 2.7 8.0 3.5 0.2 85.6Reedsgiant reed 0.0 3.4 39.2 0.3 0.8 56.33.4. Lipid composition of the fibersAs shown in Table 2, all the studied fibers present low extractives contents.However, due to the wide structural heterogeneity of the compounds that mayoccur and their different behavior during pulping, the knowledge of the chemicalnature of these components, especially the lipophilic compounds, is important inorder to predict and control the eventual pitch problems that may occur duringpulping and bleaching and to establish appropriate methods and strategies fortheir control.The composition of the lipids present in the different fibers was studied byGC and GC-MS and is shown in Table 4. The main lipid classes found in thenon-woody fibers are shown in Figure 1 and consists mainly of alkanes (A),fatty alcohols (B), aldehydes (C), fatty acids (D), sterols (G), sterol esters (H),sterol glycosides (I), steroid hydrocarbons (J), steroid ketones (K) and waxes(L). Other compounds found are alkyl ferulates (M), glycerides (N), -hydroxymonoesters (O) and -hydroxy acylesters of glycerol (P). The detailedcomposition of the lipophilic compounds present in these fibers has beenaddressed [7, 8, 22-27]. The content and composition of the different lipidclasses vary considerable among the fibers. In the case of flax bast fibers thepredominant lipophilic compounds are fatty acids and aldehydes, accounting for34% and 23% of total extract, respectively, followed by ester waxes (18%) andfatty alcohols (13%). Fatty acids are also the predominant compounds (27% oftotal extracts) in hemp bast fibers, followed by alkanes (15%), free sterols (12%)and steroid hydrocarbons (12%). The predominant lipophilic compounds in98

5. Resultados y discusiónkenaf and jute are fatty acids (28 and 35% respectively), followed by esterwaxes (26 and 27% respectively). Among the selected leaf fibers, free sterolsand fatty acids predominate in both sisal (20 and 24%, respectively) and abaca(45 and 19%, respectively), while in curaua fibers, fatty acids and ester waxespredominate (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, usuallysoda/anthraquinone pulping. Therefore, we discuss the behavior and fate of thedifferent fiber components during alkaline cooking. In this context, the lipidspresent in these fibers can be classified, in general terms, into two principalgroups, namely fatty acids (including - and -hydroxyfatty acids) and neutralcomponents, including wax esters, long-chain n-fatty alcohols, alkanes, andsteroids and triterpenoids. The different lipids classes have different behaviorduring cooking and bleaching [7, 8]. The wax esters, which are abundantlipophilic compounds in some of these fibers (i.e. flax and curaua), arehydrolyzed during alkaline cooking and the fatty acids dissolved. At sufficientlyhigh pH (as in alkaline pulping), the acids dissociate and form fatty acid soapsand can thus dissolve in water to quite a high extent. By contrast, alkanes, fattyalcohols, sterols and triterpenols, steroid hydrocarbons and ketones, and sterylglycosides do not form soluble soaps under the alkaline pulping conditions andtherefore survive cooking. These compounds have a very low solubility in waterand 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, andparticularly the low abundances of free and conjugated sterols, which have ahigh propensity to form pitch deposits [28-30] would point to a low pitchdeposition tendency of the lipophilics from these fibers. On the other hand, fattyacid soaps are effective solubilizing agents facilitating the removal from pulp ofthese sparingly soluble neutral substances. Therefore, the ratio of saponifiablesto-unsaponifiableshas been suggested to be a better index for predicting pitchproblems than the total amount of lipids (Back and Allen, 2000). In fact, thehigher abundances of unsaponifiable compounds (neutrals) with respect to thesaponifiable ones is the main cause for pitch problems during pulping of somewoods, such as aspen or eucalypt [28-31]. Fatty alcohols, alkanes and sterols areamong the compounds responsible for pitch deposits formed during pulping ofnonwoody plants [8, 32]. In most of the fibers, as in flax, hemp, kenaf or jutefibers, the content of free fatty acids (including - and -hydroxyfatty acids) ishigh, and therefore the fatty acid soaps formed during alkaline pulping maypossess sufficient micellar-forming properties to carry the less polar compoundsinto solution. However, in other fibers, such as sisal and particularly abaca, thefatty acids amounts up to only 20% of total lipophilic compounds, and thereforethey would be more prone to produce pitch deposition.99

5. Resultados y discusiónABOHOOCHDOHHOEOOOHFOHOHOHOGHOHOCH 2 OHOOHOHOIJKOOOLOOOO-CH 2OHOCH 3MNHO-O-CHHO-O-CH 2HOHOOOOOO-CH 2PHO-O-CH100HO-O-CH 2Figure 1. Structures of compounds representing the main classes of lipophilic extractivesfound in the different fibers selected. : A, pentacosane; B, docosanol; C, octacosanal; D,palmitic acid; E, 26-hydroxyhexacosanoic acid; F, 2-hydroxytetracosanoic acid; G, sitosterol;H, sitosteryl linoleate; I, sitosteryl 3-D-glucopyranoside; J, stigmasta-3,5-diene; K,stigmasta-3,5-dien-7-one; L, octacosyl hexadecanoate; M, trans-docosanylferulate; N, 1-monodocosanoylglycerol; O, docosanyl, 16-hydroxyhexadecanoate; P, 1-mono(24-hydroxytetracosanoyl)glycerol.

5. Resultados y discusiónTable 4. Composition of lipophilic extractives (mg/100g) in the different fibers [8, 22-26].Bast fibers Leaf fibers Reedsflax hemp kenaf jute sisal abaca curaua giant reedn-alkanes (A) 27 43 27 5 15 - - 8fatty alcohols (B) 220 2 13 13 8

5. Resultados y discusiónfor the non-woody fibers studied here is listed in Table 5. A predominance of S-over G-lignin was found in the bast fibers of kenaf [24] and jute [20, 36] and inall the leaf fibers of sisal, abaca and curaua [20, 22, 23]. By contrast, the bastfibers from flax and hemp, as well as the giant reed showed a predominance ofG-lignin [20, 21, 26]. This is especially evident in the lignin of flax, with anextremely low S/G ratio of 0.1. The low S/G ratio of the lignins from flax andhemp, despite having very low lignin contents (less than 5% Klason lignin),makes them fairly resistant to alkaline delignification. Also, the high lignincontent of giant reed (24.7 % Klason lignin) together with its low S/G ratio of0.7 makes it especially hard to delignify. By contrast, the rest of the fibers(kenaf, jute, sisal, abaca, curaua) present both high S/G ratios and low lignincontents, which will make them easily delignifiable under alkaline pulping,requiring lower energy and less drastic conditions.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 Reedsflax 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 30.2S/G ratio 0.1 0.6 5.4 2.0 4.3 4.9 1.4 0.7For a more complete structural characterization of the lignins from these nonwoodyfibers, the milled-wood lignins (MWL) were isolated and analyzed by2D-NMR spectroscopy [36-38]. Signals from S and G lignin units wereobserved in all spectra, whereas signals for p-hydroxyphenyl (H) lignin unitscould only be detected in the HSQC spectra of flax and hemp lignins, inagreement with the high amounts of these units observed by Py-GC/MS andshown in Table 5. In general, the relative proportions of the different ligninunits (S/G ratios in Table 6) are in close agreement with the Py-GC/MS datashown above. In addition, prominent signals corresponding to p-coumaratestructures were observed in the lignins of abaca and curaua [37]. In theselignins, 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 additionalinformation about the different inter-unit linkages (i.e. -O-4 aryl ether, -resinol, -5 phenylcoumaran, -1/-O- spirodienones, etc) present in thestructure of these lignins. The main substructures found in these lignins aredepicted in Figure 2. The relative abundances of the main inter-unit linkagespresent in the MWL of the non-woody fibers selected for this study are shown inTable 6. -O-4 aryl ether substructures (I) were predominant in all of thelignins. Interestingly, the lignins from kenaf, sisal, abaca and curaua are102

5. Resultados y discusiónespecially enriched in -O-4 structures (more than 84% of all side-chains) [37].Phenylcoumaran (-5 linkages) substructures (II) were observed in most of thefibers, being especially abundant in flax and hemp, but were completely absentin abaca. The presence of these low amounts of phenylcoumaran substructureswas expected due to the low levels of guaiacyl lignin units in these samples.Resinol (- linkages) substructures (III) were also observed in importantamounts in flax, hemp, and jute, and in low amounts in kenaf and sisal, but werecompletely absent in abaca and curaua lignins. Finally, spirodienone structures(IV) were also present, although in lower amounts in most of the fibers, beingabsent in flax and hemp. The high abundance of non-condensed linkages in thelignins of kenaf, sisal, abaca and curaua makes them particularly easily todelignify, in contrast to the rest of the lignins, with a high content of condensedlinkages, particularly in flax and hemp lignins.Table 6. Structural characteristics (relative abundance of the main interunit linkages aspercentages of side-chains involved, percentage of -acylation and S/G ratio) observed fromthe HSQC spectra of the MWL of selected fibers (curaua, hemp, kenaf, jute, sisal and abaca)[35-37].Bast fibersLeaf fibersflax hemp kenaf jute sisal abaca curauaLinkage relative abundance (% of side-chains involved)-O-4 alkyl-aryl units (I, I', I'') 71 69 84 72 89 94 94Phenylcoumarans (II) 16 9 2 4 2 0 2Resinols (III) 13 22 8 16 4 0 0Spirodienones (IV) 0 0 6 4 5 6 4Percentage of -acylation 0 0 58 4 68 80 69S/G ratio 0.1 0.8 5.6 2.0 3.9 8.7 4.9Interestingly, 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 signalscorresponding to the hydroxylated and acylated -carbon (Table 6) and rangedfrom 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 andthe low abundances of the - resinol structures [37, 38]. The nature of the acylgroup esterifying the -carbon was studied by the so-called DerivatizationFollowed by Reductive Cleavage (DFRC) degradation method [40, 41], whichselectively and efficiently cleaves -ether and -ether linkages but leaves -esters intact. This method allowed confirming that p-coumarate groups are103

5. Resultados y discusiónattached at the -carbon of abaca and curaua lignins, and predominantly onsyringyl units [37, 41]. In addition, acetate units were also found esterifying the-carbon in the lignins of all the studied fibers, although at different extents. Inall cases, acetate and p-coumarate groups were found to be preferentiallyattached to syringyl units [37, 41-43]. It must be noted that, although these estermoieties will, in principle, consume additional alkaline reagents during cooking,it has been shown above that the highly acylated lignins are extremely enrichedin easily hydrolysable non-condensed -O-4 linkages, which will be moreamenable to delignification.HO4´5´6´O3´1´2´´´´OHOHOMeOO5’4’6’1’2’3’HOOMeOO5’4’6’1’2’3’HOOMeOO5'’4'’6'’1'’2'’3'’MeO651423OMeOMeMeO651423OMeOMeMeO651423OMeOMeOOOII'I''HOMeO651’6’ 2’5’ 3’4’OMe O1234OMeOMeOO546123OMeOO’’’OMe3’2’ 4’1’ 5’6’OOMeOMeO1''6'' 2''343''25''4''5MeO1 MeO6O OOMe ´´HO´1´OH6´5´2´3´MeO4´OMeOIIIIIFigure 2. Main substructures present in the lignins studied here: I, -O-4 linkedsubstructures; I, -O-4 linked substructures with acetylated -carbon; I, -O-4 linkedsubstructures; with p-coumaroylated -carbon; II, phenylcoumaran structures formed by -5and -O-4 linkages; III, resinol structures formed by - , -O-, and -O-R linkages; IV,spirodienone structures formed by -1, and -O- linkages.IV4. ConclusionsThe chemical composition of different non-woody plant fibers used as rawmaterials for pulp and papermaking has been summarized, with especialemphasis in the chemistry of lipids and lignin and their fate during alkaline104

5. Resultados y discusiónpulping. This study offers valuable information that will lead to a betterindustrial utilization of these non-woody plant species of high socioeconomicinterest.AcknowledgementsThis study has been supported by the Spanish Projects AGL2005-01748 andAGL2008-00709 and the EU BIORENEW project (NMP2-CT-2006-26456).We thank CELESA (Tortosa, Spain) and University of Huelva for providing thesamples. 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 Int1998; 61-86.[3] Saijonkari-Pahkala K. Non-wood plants as raw materials for pulp andpaper. PhD Thesis, University of Helsinki, Finland, 2001; pp. 101.[4] Hurter RW, Riccio FA. Why CEOS don’t want to hear about nonwoods-orshould they? In: TAPPI Proceeding, NA Nonwood Fiber Symposium,Atlanta, GA, USA, 1998; 1-11.[5] del Río JC, Gutiérrez A, Hernando M, Landín P, Romero J, Martínez AT.Determining the influence of eucalypt lignin composition in paper pulpyield using Py-GC/MS. J Anal Appl Pyrol 2005; 74: 110-115.[6] Back EL, Allen LH. Pitch Control, Wood Resin and Deresination, Tappipress, Atlanta, GA., 2000; pp. 392.[7] Gutiérrez A, del Río JC. Lipids from flax fibers and their fate after alkalinepulping. J Agric Food Chem 2003; 51: 4965-4971.[8] Marques G, del Río JC, Gutiérrez A. Lipophilic extractives from severalnonwoody lignocellulosic crops (flax, hemp, sisal, abaca) and their fateduring alkaline pulping and TCF/ECF bleaching. Biores Technol 2010;101: 260-267.[9] Technical Association of the Pulp and Paper Industry. Test methods, 1992-1993. TAPPI, Atlanta, Ga. 1993.105

5. Resultados y discusión[10] Gutiérrez A, del Río JC, González-Vila FJ, Martín F. Analysis of lipophilicextractives from wood and pitch deposits by solid-phase extraction and gaschromatography. J Chromatogr A 1998; 823: 449-455.[11] Christie WW. In Christie WW, Ed. Solid-phase extraction columns in theanalysis of lipids. Advances in Lipid Methodology—One, The Oily Press,Dundee, Scotland 1992; pp. 1–18.[12] Faix O, Meier D, Fortmann I. Thermal degradation products of wood. Acollection of electron of electron-impact (EI) mass spectra of monomericlignin derived products. Holz Roh- Werkst 1990; 48 (9): 351-354.[13] Ralph J, Hatfield RD. Pyrolysis-GC/MS characterization of foragematerials. J Agric Food Chem 1991; 39 (8): 1426-1437.[14] Björkman A. Studies on finely divided wood. Part I. Extraction of ligninwith neutral solvents. Sven Papperstidn 1956; 59: 477-485.[15] Capanema EA, Balakshin MY, Chen CL, Gratzl JS, Gracz H. Structuralanalysis of residual and technical lignins by 1 H- 13 C correlation 2D NMRspectroscopy.Holzforschung 2001; 55: 302-308.[16] Capanema EA, Balakshin MY, Kadla JF. Quantitative characterization of ahardwood milled wood lignin by nuclear magnetic resonance spectroscopy.J Agric Food Chem 2005; 53 (25): 9639–9649.[17] Liitiä TM, Maunu SL, Hortling B, Toikka M, Kilpeläinen I. Analysis oftechnical lignins by two- and three-dimensional NMR spectroscopy. JAgric Food Chem 2003; 51 (21): 2136–2143.[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, BoudetAM. Solution-state NMR of lignin. In: Argyropoulos DS, Ed. Advances inLignocellulosics Characterization, Tappi Press, Atlanta, GA 1999; 55-108.[19] García Hortal JA. Fibras Papeleras, Edicions UPC, Barcelona, Spain, 2007;pp. 243.[20] Rodríguez MI. Caracterización química de plantas hebáceas utilizadas parala fabricación de pasta de papel de alta calidad. PhD Thesis, University ofSeville, Spain 2006.106

5. Resultados y discusión[21] Coelho DS Estudo sistemático da composiçao quimica das fibras deArundo donax e a sua evoluçao durante a producto de pasta de papelatravés do processo organosolv. MSc Thesis, University of Aveiro,Portugal, 2006.[22] Marques G, Gutiérrez A, del Río JC. Chemical characterization of ligninand lipophilic fractions from leaf fibers of curaua (Ananas erectifolius). JAgric Food Chem 2007; 55: 1327-1336.[23] del Río JC, Gutiérrez A. Chemical composition of abaca (Musa textilis)leaf fibers used for manufacturing of high quality paper pulps. J Agric FoodChem 2006; 54 (13): 4600–4610.[24] Gutiérrez A., Rodríguez IM, del Río JC. Chemical characterization oflignin and lipid fractions in kenaf bast fibers used for manufacturing highqualitypapers. J Agric Food Chem 2004¸ 52: 4764-4773.[25] del Río JC, Marques G, Rodríguez IM, Gutiérrez A. Chemical compositionof lipophilic extractives from jute (Corchorus capsularis) fibers used formanufacturing of high-quality paper pulps. Ind Crops Prod 2009; 30: 241-249.[26] Gutiérrez A, Rodríguez IM, del Río JC. Chemical characterization of ligninand lipid fractions in industrial hemp bast fibers used for manufacturinghigh-quality paper pulps. J Agric Food Chem 2006; 54: 2138-2144.[27] Coelho DS, Marques G, Gutiérrez A, Silvestre ARD, del Río JC. Chemicalcharacterization of the lipophilic fraction of Giant reed (Arundo donax)fibres used for pulp and paper manufacturing. Ind Crops Prod 2007; 26:229-236.[28] del Río JC, Gutiérrez A, González-Vila FC, Martín F, Romero J.Characterization of organic deposits produced in kraft pulping ofEucalyptus globulus wood. J Chromatogr A 1998; 823: 457–465.[29] del Río JC, Romero J, Gutiérrez A. Analysis of pitch deposits produced inkraft pulp mills using a totally chlorine free bleaching sequence. JChromatogr A 2000; 874: 235–245.[30] Gutiérrez A, del Río JC. Gas chromatography/mass spectrometrydemonstration of steryl glycosides in eucalypt wood, kraft pulp and processliquids. Rapid Commun Mass Spectrom 2001; 15: 2515–2520.[31] Chen T, Wang Z, Zhou Y, Breui, C, Aschim OK, Yee E, Nadeau L. Usingsolid-phase extraction to assess why aspen causes more pitch problemsthan softwoods in kraft pulping. Tappi J 1995; 78: 143-149.107

5. Resultados y discusión[32] Gutiérrez A, del Río JC. Chemical characterization of pitch depositsproduced in the manufacturing of high-quality paper pulps from hempfibers. Biores Technol 2005; 96: 1445-1450.[33] González-Vila FJ, Almendros G, del Río JC, Martín F, Gutiérrez A,Romero J. Ease of delignification assessment of wood from differentEucalyptus species by pyrolysis (TMAH)-GC/MS and CP/MAS 13 C NMRspectrometry. J Anal Appl Pyrol 1999; 49: 295-305.[34] Chang HM, Sarkanen KV. Species variation in lignin. Effect of species onthe rate of Kraft delignification. Tappi press 1973; 56: 132–134.[35] Tsutsumi Y, Kondo R, Sakai K, Imamura H. The difference of reactivitybetween syringyl lignin and guaiacyl lignin in alkaline systems.Holzforschung 1995; 49 (5): 423-428.[36] del Río JC, Rencoret J, Marques G, Li J, Gellerstedt G, Jiménez-Barbero J,Martínez AT, Gutiérrez A. Structural characterization of the lignin fromjute (Corchorus capsularis) fibers. J Agric Food Chem 2009; 57: 10271-10281.[37] del Río JC, Rencoret J, Marques G, Gutiérrez A., Ibarra D, Santos JI,Jiménez-Barbero J, Zhang L, Martínez AT. Highly acylated (acetylatedand/or p-coumaroylated) native lignins from diverse herbaceous plants. JAgric Food Chem 2008; 56: 9525-9534.[38] Martínez AT, Rencoret J, Marques G, Gutiérrez A, Ibarra D, Jiménez-Barbero J, del Río JC. Monolignol acylation and lignin structure in somenonwoody plants: A 2D NMR study. Phytochemistry 2008; 69: 2831-2843.[39] Sun RC, Fang JM, Goodwin A, Lawther JM, Bolton J. Fractionation andcharacterization of ball-milled and enzyme lignins from abaca fibre. J SciFood Agric 1999; 79: 1091-1098.[40] Lu F, Ralph J. Derivatization followed by reductive cleavage (DFRCmethod), a new method for lignin analysis: protocol for analysis of DFRCmonomers. J Agric Food Chem 1997; 45 (7): 2590-2592.[41] del Río JC, Marques G, Rencoret J, Martínez AT, Gutiérrez A. Occurrenceof naturally acetylated lignin units. J Agric Food Chem 2007; 55:5461-5468.[42] Ralph J, Lu F. The DFRC method for lignin analysis. 6. A simplemodification for identifying natural acetates in lignin. J Agric Food Chem1998; 46: 4616-4619.108

5. Resultados y discusión[43] Ralph J. An unusual lignin from kenaf. J Nat Prod 1996; 59 (4): 341-342.109

5. Resultados y discusiónPublicación II:del Río J.C., Marques G., Rencoret J. Martínez A.T. and Gutiérrez A. (2007)Occurence of naturally acetylated lignin units. Journal of Agricultural and FoodChemistry, 55, 5461-5468.110

Occurrence of naturally acetylated lignin units5. Resultados y discusiónJosé C. del Río † , Gisela Marques † , Jorge Rencoret † , Ángel T. Martínez ‡ , Ana Gutiérrez †† Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, P.O. Box 1052, 41080-Seville, Spain‡ Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, SpainAbstractIn this work, we have studied the occurrence of native acetylated lignin in alarge set of vascular plants, including both angiosperms and gymnosperms, by amodification of the so-called Derivatization Followed by Reductive Cleavage(DFRC) method. Acetylated lignin units were found in all angiosperms selectedfor this study, including mono- and eudicotyledons, but were absent in thegymnosperms analyzed. In some plants (e.g. abaca, sisal, kenaf or hornbeam),lignin acetylation occurred at a very high extent, exceeding 45% of theuncondensed syringyl lignin units. Acetylation was observed exclusively at the-carbon of the lignin-side chain and predominantly on syringyl units, althougha predominance of acetylated guaiacyl over syringyl units was observed in someplants. In all cases, acetylation appears to occur at the monomer stage andsinapyl and coniferyl acetates seem to behave as real lignin monomersparticipating in lignification.Keywords: lignin, angiosperms, gymnosperms, eudicotyledons,monocotyledons, coniferyl acetate, sinapyl acetate, abaca, sisal, kenaf,hornbeam, Derivatization Followed by Reductive Cleavage (DFRC).1. IntroductionLignin is a principal structural component of cell walls in higher terrestrialplants. In addition to structural support and pathogen defense, lignin functions inwater transport as a hydrophobic constituent of vascular phloem and xylem cells.Lignins are complex polymers formed by the dehydrogenative polymerization ofthree main monolignols, p-coumaryl, coniferyl and sinapyl alcohols.Gymnosperm lignins are mainly formed from coniferyl alcohol, together withsmall proportions of p-coumaryl alcohol. Angiosperm lignins are mainly formedfrom coniferyl and sinapyl alcohols with small amounts of p-coumaryl alcohol.A considerable variation in the contribution of these three alcohol precursors isobserved in lignins from annual plants (1-4). After their synthesis, the ligninmonomers are transported to the cell wall where they are polymerized in acombinatorial fashion by free-radical coupling mechanisms in a reactionmediated by peroxidases, generating a variety of structures within the ligninpolymer (5-7).111

5. Resultados y discusiónSome lignins are known to be naturally acetylated at the -carbon of the sidechain. Acetates have been reported to occur in the lignin of a limited number ofhardwoods and non-woody plants (1, 8, 9). In particular, kenaf bast lignin hasbeen found to be extensively -acetylated and predominantly on syringyl units,although the role of such a highly acetylated lignin is not yet known. Recentstudies have provided strong evidence that sinapyl acetate is implicated as amonomer in lignification in kenaf bast fibers and that the naturally acetylatedpolymeric lignin derives not from acetylation of the lignin polymer but frompolymerization of pre-acetylated monolignols (9, 10). Other acids are alsoknown to occur naturally acylating lignin; thus, grass lignins are partially p-coumaroylated and some hardwood lignins such as poplar, aspen or willow arep-hydroxybenzoylated (7, 11-18).Naturally acetylated lignins may also occur in other plants but theiroccurrence has probably being missed due to the limitations of the analyticalprocedures used for their isolation and/or structural characterization. Naturalacetates present on lignin might have been hydrolyzed and removed when usingtraditional isolation methods (such as alkaline extraction often applied to nonwoodlignins) and degradative procedures for chemical characterization (such asnitrobenzene oxidation, CuO oxidation or thioacidolysis). Indeed, forspectroscopic analysis, e.g. using Nuclear Magnetic Resonance (NMR), lignin isfrequently acetylated for improved solubility and spectroscopic properties and,therefore, native acetates cannot be seen. Recently, we reported the occurrenceof acetylated lignins in some herbaceous plants, including kenaf, jute, sisal andabaca, characterized by a high syringyl (S) to guaiacyl (G) ratio, by the use ofPyrolysis coupled to Gas Chromatography-Mass Spectrometry (Py-GC/MS)(19-21), although the method used hindered the determination of the extent ofacetylation. In this work we have studied the occurrence and abundance ofnative acetylated lignin units in the milled wood lignins (MWL) isolated from awide set of vascular plants, including gymnosperms and angiosperms (monoandeudicotyledons). For this purpose, we have used a modification of the socalledDerivatization Followed by Reductive Cleavage (DFRC) degradationmethod (22-24). DFRC is a simple and powerful method which selectively andefficiently cleaves -ether and -ether linkages and allows quantitative analysisof structural units in etherified lignin, and also provides some information oncarbon-carbon linked lignin by analysis of the dimeric structures released.DFRC includes two key steps, (i) solubilization of lignin by bromination andacetylation with acetyl bromide and (ii) reductive cleavage of the aryl etherbonds in lignin with zinc dust. Identification of the resulting monomeric anddimeric degradation products by GC/MS gives valuable information on thelignin structure. However, and most importantly, DFRC leaves -esters intactallowing the analysis of native -acylated lignin. Thus, the method has allowedto confirm that p-coumarate groups are attached at the -carbon of grass lignins,predominantly on syringyl units (17). However, the DFRC method uses112

5. Resultados y discusiónacetylating reagents that interfere in the analysis of native acetates in lignin, butwith appropriate modification by substituting acetylation by propionylation (25),it is possible to obtain significant information and clues about the occurrenceand extent of native lignin acetylation. In this paper, we use this method toinvestigate the presence of naturally acetylated lignin units in a set of vascularplants, including angiosperms and gymnosperms.2. Material and methods2.1. SamplesThe plant samples selected for this study are listed in Table 1. They consist ofboth woody and nonwoody angiosperms (mono- and eudicotyledons) andgymnosperms. Among the woody angiosperms, wood of beech (Fagussylvatica), European hornbeam (Carpinus betulus), aspen (Populus tremula),and eucalyptus (Eucalyptus globulus) were selected. The nonwoody angiospermsamples consisted of bast fibers obtained from the stalk phloem layer of bamboo(Bambusa sp.), hemp (Cannabis sativa), kenaf (Hibiscus cannabinus) and jute(Corchorus capsularis); leaf fibers of sisal (Agave sisalana) and abaca (Musatextilis); and coir, a coarse fiber obtained from the outer shell of coconut fromthe palm tree (Cocos nucifera). Among gymnosperm woods, Scots pine (Pinussylvestris) and Norway spruce (Picea abies) were selected for this study. Woodand nonwoody plants were finely ground to sawdust using a knife mill(Analysenmühle A10, Janke and Kunkel GmbH, Staufen, Germany) beforeanalysis. MWL was extracted from finely ball-milled (150 h) plant material, freeof extractives and hot water soluble material, using dioxane-water (9:1, v/v),followed by evaporation of the solvent, and purified as described (26). The finalyields ranged from 5-15% of the original lignin content. Extension of millingtime, which would increase yield, was avoided in order to prevent chemicalmodifications on the lignin structure.2.2. DFRCA modification of the standard DFRC method by using propionyl instead ofacetyl reagents (DFRC´) was used (25). Lignins (10 mg) were stirred for twohours at 50 ºC with propionyl bromide in propionic acid (8:92, v/v). Thesolvents and excess of bromide were removed by rotary evaporation. Theproducts were then dissolved in dioxane/propionic acid/water (5:4:1, v/v/v), and50 mg powered Zn was added. After 40 min stirring at room temperature, themixture was transferred into a separatory funnel with dichloromethane andsaturated ammonium chloride. The aqueous phase was adjusted to pH < 3 byadding 3% HCl, the mixture vigorously mixed and the organic layer separated.The water phase was extracted twice more with dichloromethane. The combineddichloromethane fractions were dried over anhydrous NaSO 4 and the filtrate wasevaporated in a rotary evaporator. The residue was subsequently propionylated113

5. Resultados y discusiónfor 1 h in 1.1 mL of dichloromethane containing 0.2 mL of propionic anhydrideand 0.2 mL pyridine. The propionylated lignin degradation compounds werecollected after rotary evaporation of the solvents, and subsequently analyzed byGC/MS.2.3. GC/MS analysisThe GC/MS analyses were performed with a Star 3400 GC (Varian) equippedwith a Saturn 2000 ion trap detector (Varian) using a 12m x 0.25 mm i.d., 0.1m, DB-5HT length capillary column (J&W Scientific, Folsom, CA, USA). Theoven was heated from 50 (held 0.2 min) to 100 ºC at 30 ºC/min, then raised to300 ºC at 5ºC/min, and held for 5 min at the final temperature. The injector andtransfer line were kept at 300 ºC. Helium was used as the carrier gas at a rate of2 mL/min.Quantification of the released individual monomers was performed usingtetracosane as external standard and by assuming similar response factors asthose of the acetylated monomers reported in Lu and Ralph (22), althoughwithout authentication on our instrument. Molar yields were calculated on thebasis of molecular weights of the respective propionylated (or acetylated)compounds.3. Results and discussionThe MWL isolated from the different vascular plants selected for this study,including angiosperms (mono- and eudicotyledons) and gymnosperms, wereanalyzed by the modified DFRC´ method developed by Lu and Ralph (25) inorder to investigate the occurrence of naturally acetylated lignin moieties.However, we have to emphasize here that the monomeric degradation productsreleased by DFRC´ originate from etherified lignin units since this method onlycleaves - and -aryl ether bonds. The abundance of guaiacyl lignin units,which are predominantly in condensed form (i.e. forming 5-5´ linkages), mayalso be underestimated.The chromatograms of the DFRC´ degradation products of selected MWLsamples are shown in Figure 1. All the analyzed lignins released the cis andtrans isomers of guaiacyl (c-G and t-G) and syringyl (c-S and t-S) ligninmonomers (as their propionylated derivatives) in different proportions, arisingfrom normal (-OH) units in lignin. In addition, the presence of originally -acetylated guaiacyl (c-G ac and t-G ac ) and syringyl (c-S ac and t-S ac ) lignin unitscould also be clearly observed in the chromatograms of most of the analyzedlignins. The structures and mass fragments of these compounds arising from -OH and from -acetylated lignin units are depicted in Figure 2. Acetylationoccurred exclusively at the -carbon of the lignin side-chain, as already reportedfor kenaf lignin (1, 25). In all samples, coniferyl and sinapyl acetates presented a114

5. Resultados y discusiónTable 1. Abundance (Molar Yields) of the DFRC´ Degradation Monomers of the MWL Isolated from the Different Plants Selected forThis Study, S/G Ratios and Relative Abundances of Acetylated Lignin Moieties.Monomers (mol/g lignin)Order Family Species Name G Gac S Sac S/G %Sac a %Gac bAngiospermsMonocotyledonsAsparagales Agavaceae Agave sisalana Sisal 122 124 108 378 2.0 77.7 50.4Arecales Arecaceae Cocos nucifera Coir 819 5 174 14 0.2 7.4 0.6Poales Poaceae Bambusa sp. Bamboo 256 13 280 4 1.1 c 1.2 c 4.8Zingiberales Musaceae Musa textilis Abaca 50 3 21 131 3.0 c 80.3 c 5.6EudicotyledonsFagales Fagaceae Fagus sylvatica Beech 126 2 165 20 1.4 10.8 1.6Fagales Betulaceae Carpinus betulus Hornbeam 146 4 230 185 2.8 44.6 2.7Rosales Cannabaceae Cannabis sativa Hemp 286 2 177 2 0.6 1.1 0.7Malvales Malvaceae Hibiscus cannabinus Kenaf 390 38 543 780 3.1 59.0 8.9Malvales Malvaceae Corchorus capsularis Jute 299 1 336 23 1.2 6.4 0.3Malpighiales Salicaceae Populus tremula Aspen 651 5 662 8 1.0 1.2 0.8Myrtales Myrtaceae Eucalyptus globulus Eucalypt 154 8 275 3 2.3 1.1 4.9GymnospermsConiferales Pinaceae Picea abies Spruce 520 0 0 0 0.0 - 0.0Coniferales PinaceaePinus sylvestris Pine 402 0 0 0 0.0 - 0.0%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. c Some amounts of-p-coumaroylated S units were found (27 an 11 mol/g lignin for bamboo and abaca, respectively) and were included in the estimation of total S units for calculationaof S/G and %Sac.115

5. Resultados y discusiónpredominance of the trans- over the cis- form, as also occurred with thecorresponding 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 thepercentages of naturally acetylated guaiacyl (%G ac ) and syringyl (%S ac ) ligninmoieties, are presented in Table 1. The yields of the released monomers were inthe same range as previously observed in the DFRC degradation of otherisolated lignins (17, 24). As shown in Table 1, naturally acetylated lignin unitswere found to occur in all angiosperms analyzed in the present study, includingboth mono- and eudicotyledons. However, no traces of acetylated lignin unitscould be found in the MWL of the two gymnosperms (pine and spruce) studiedhere. The data also indicated that in most lignin samples acetylation occurredpredominantly on syringyl units, whereas only traces of acetylated guaiacylunits were detected, although in bamboo and eucalyptus lignins, with a lowextent of acetylation, this occurred preferentially on guaiacyl units. We canexclude acetylation as an artifact produced during the lignin isolation protocolsince MWL from pine and spruce (where no traces of acetylated units could bedetected) were also isolated using the same procedure as the rest of the samples.Indeed, acetates were found predominantly on S lignin units and exclusively atthe -carbon, which suggests that they are naturally present. And finally, directDFRC´ of some whole fibers (such as sisal and kenaf), without previous ligninisolation, gave also similar results.The occurrence of naturally acetylated lignin units seems to be widespreadamong angiosperms and restricted only to this group of vascular plants, beingparticularly abundant in syringyl-rich lignins. Especially important is the highextent of lignin acetylation observed in the MWL from the herbaceous plantsabaca, 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 theacetylation of S units in coir lignin, which is characterized by a very low S/Gratio (0.2). The high extent of acetylation of kenaf lignin has been previouslyreported by NMR and DFRC´ (1, 25). The occurrence of naturally acetylatedlignin units was also reported in kenaf, jute, sisal and abaca by Py-GC/MS of thewhole fibers, although the extent of acetylation could not be determined due tothe limitations of the technique (19-21). Interestingly, the high extent ofacetylation of sisal MWL included both S units (78%) and G units (50%),whereas in the case of abaca, kenaf or hornbeam lignins, acetylation occurredalmost predominantly on S units (80, 59 and 45%, respectively) and only aminor degree of acetylation was observed on G units (6, 9 and 3%, respectively).116

5. Resultados y discusión100Sisalrelative intensity0100relative intensity0100relative intensityt-S act-St-Gt-G acc-Gc-S ac c-Sc-G acc-Gact-G act-S act-St-Gc-G c-S acc-St-Gc-G t-Gacc-S acc-St-S act-SKenafHornbeam100t-S ac0relative intensityAbacat-Gt-Sc-Gc-S act-G acc-S010 15 20Retention 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 syringylmonomers, respectively. c-G ac , t-G ac , c-S ac and t-S ac are the originally acetylated cis- andtrans-guaiacyl and syringyl monomers, respectively.On the other hand, it has been recently and elegantly proved that acetylatedlignin in kenaf derives from polymerization of pre-acetylated monolignols andnot from acetylation of the lignin polymer (9, 10). Acetylation of lignin atmonomer stage only partially affects the course of the coupling reactions, and117

5. Resultados y discusiónthese acetylated monolignols can still undergo the O–4-coupling reactionsthat incorporate them into the lignin polymer. Obviously, the resultingacetylated lignin polymer is produced and has the mechanical propertiesrequired by the plant. However, the presence of -acetylated monolignols altersto some extent the structure of the lignins because the -OH group of amonolignol participates in some post-coupling reactions, such as coupling,internally trapping the quinone methide. With the -OH group acetylated, suchinternal reactions are no longer possible and the quinone methide must berearomatized by trapping an external nucleophile, usually water, and forming asa result new products in the lignin different to the expected resinolstructures formed from non-acetylated monolignols (9). Figure 3 shows thedifferent tetrahydrofuran structures arising from the homo- and crosscouplingof the two sinapyl (acetylated and non-acetylated) monolignols. It isclear that structures I and II can only be formed if sinapyl alcohol is preacetylatedand then undergoes coupling. These homo- (I) and crosscoupled(II) structures arising from pre-acetylated sinapyl alcohol were found inkenaf lignin by DFRC´ and 2D-NMR (9, 10), which unequivocally demonstratesthat sinapyl alcohol is pre-acetylated prior to lignification and that sinapylacetate behaves as a real monolignol in kenaf lignin.OPropOPropOCH 3OPropCH 3 OOCH 3OPropGM + = 292; [M + -56]= 236SM + = 322; [M + -56]= 266OO-C-CH 3=OO-C-CH 3=OCH 3OPropCH 3 OOCH 3OPropG acM + = 278; [M + -56]= 222S acM + = 308; [M + -56]= 252Figure 2. Structures and mass fragments of the -OH (G and S) and naturally -acetylated(G ac and S ac ) lignin monomers released after DFRC´ of MWL.118

5. Resultados y discusiónOOCH 3 OOH=O-C-CH 3+OCH 3 CH 3 OOH=O-C-CH 3OCH 3homo-couplingO=CH 3 -C-OCH 3 OHOOCH 3OIO=O-C-CH 3OCH 3OHOCH 3OHOCH 3 OOCH 3OH=O-C-CH 3CH 3 OOH+OCH 3 CH 3 OOHOCH 3cross-couplingHO O=CH 3 -C-OOOCH 3CH 3 OIIOHOCH 3OHCH 3 OOHOH+OCH 3 CH 3 OOHOHOCH 3homo-couplingCH 3 OHOOCH 3OOIIIOCH 3Figure 3. Structures of the tetrahydrofuran dimers arising from the coupling of sinapylalcohol and sinapyl acetate. I: coupling product of two sinapyl acetates; II: couplingproduct of a sinapyl alcohol and a sinapyl acetate; III: coupling product (syringaresinol)of two sinapyl alcohols.We also investigated the presence of the tetrahydrofuran structures arisingfrom coupling of sinapyl acetates in the MWL selected for this study, byusing the DFRC´ method. The DFRC´ degradation products of thetetrahydrofuran structures depicted in Figure 3 are presented in Figure 4, andtheir mass spectra have already been published (9). Compounds I´, II´a and II´b,arising from originally acetylated sinapyl alcohol, together with compound III´arising from the resinol structure, were found in most of the samples analyzed.Figure 5 shows the chromatograms (sum of the single ion chromatograms of therespective base peaks) of the DFRC´ degradation products of the tetrahydrofuran119

5. Resultados y discusiónstructures arising from coupling of sinapyl alcohol (and its acetylatedcounterpart) in selected samples. It is interesting to note the relatively highamounts of compounds I´, II´a and II´b, arising from native acetylated ligninunits in kenaf and especially in sisal lignin and the complete absence of coupling structures in abaca lignin. However, we also observed that therespective tetrahydrofuran structures arising from the coupling of theacetylated guaiacyl counterparts could not be found, even in the case of sisallignin (with 50% acetylated ether-linked G units). The presence of compoundI´(arising from structure I in lignin) and compounds II´a and II´b (arising fromstructure II) in the DFRC´ degradation products of sisal, kenaf, jute, hornbeam,and other lignins clearly indicates that in these samples sinapyl alcohol is preacetylatedand behave as a real monolignols participating in post cross-couplingreactions. Therefore, it is possible that in all angiosperms, in which we haveshown the occurrence of acetylated lignin units, sinapyl and possibly coniferylacetates participate in lignification as true lignin precursors. This implies that thenaturally acetylated polymeric lignins in all these plants derive not fromacetylation of the lignin polymer but from polymerization of pre-acetylatedmonolignols, as already suggested (9). Thus, the traditional concepts of bothlignin biosynthesis and structure must be reconsidered. This indicates, inagreement with other authors (7, 27) that the lignification process is veryflexible, and that the definition of lignin must not be restricted to a polymer ofthe three traditional hydroxycinnamyl alcohols.The relative abundance of the compounds released in Figure 5 gives someadditional information. In sisal, the relative molar abundance of the acetylatedversus the non-acetylated sinapyl alcohols forming linkages is 44:56, with apredominance of the non-acetylated sinapyl alcohol, whereas their relative molarabundances in ether-linked structures is 78:22, with a strong predominance ofsinapyl acetate. This indicates that sinapyl acetate has a lower affinity to form linkages than the normal sinapyl alcohol and therefore those lignins havinga high extent of acetylation would produce lower amounts of linkages. Thisis in agreement with the high proportions of -O-4 substructures present in sisallignin, as indicated by 2D-NMR (data not shown). Interestingly, abaca MWL,with a very high extent of acetylation (80% of S units), lacks linkages,including those arising from normal non-acetylated lignin units (Figure 5), andproduces almost exclusively O-4 substructures. Similar results were observedafter normal DFRC and 2D-NMR (data not shown). Therefore, it seems that thehigh extent of -acetylation would favor the formation of a predominantlylignin structure. The difficulty in finding substructures arising fromnaturally acetylated guaiacyl lignin in sisal, despite the high abundance of thistype of units, would be perhaps due to the low amounts of linkagesproduced.120

5. Resultados y discusiónCH 3 OO=O-C-CH 3IDFRC´PrOCH 3 OO-C-CH 3=OCH 3 OOCH 3OPrI´; M + =616; [M-56] + = 560CH 3 OOPrCH 3 OO=O-C-CH 3IIDFRC´PrOCH 3 OO-C-CH 3=O+PrOCH 3 OOPrCH 3 OOCH 3CH 3 OOCH 3OPrII´a; M + = 630; [M-56] + = 574OPrII´b; M + = 630: [M-56] + = 574CH 3 OOPrIIIDFRC´PrOCH 3 OOPrCH 3 OOCH 3OPrIII´; M + = 644; [M-56] + = 588Figure 4. Aryltetralin products resulting from the DFRC´ degradation of the di- (I´), mono-(II´a and II´b) and none-acetylated (III´) coupling structures. The molecular mass andbase 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 inthe plant is not yet known. Some studies indicated that -acylation with p-coumarates may function as radical transfer carriers to help sinapyl alcoholincorporate into lignin when the wall peroxidases have a low reactivity withsinapyl alcohol directly (28). However, this is not the case for acetylated ligninmonomers and the function of such acetylated lignin remains unknown.121

5. Resultados y discusión100II´a,II´bIII´Sisalrelative intensityI´0100III´Kenafrelative intensityI´II´a,II´b0100III´Hornbeamrelative intensityII´a,II´b0100Abacarelative intensity035 36 37 38 39 40Retention time (min)Figure 5. Detail of reconstructed (sum of the ions at m/z 560, 574 and 588) chromatograms ofthe DFRC´ degradation products of selected MWL from sisal, kenaf, hornbeam and abaca,showing the presence of aryltetralin products containing two (I´), one (IIá and II´b) andnone (III´) native acetates.The high extent of lignin acetylation of sisal and other Agaves (preliminaryresults on another species, Agave americana, by DFRC´ of the whole fiber,without previous lignin isolation, also indicate more than 75% of acetylated Sunits) can provide some answers about its role in this plant. Since the resultant122

5. Resultados y discusiónacetylated lignin polymer is more hydrophobic than normal lignin, the role oflignin acetylation could be associated with drought tolerance, as alreadyadvanced by Ralph (29). Sisal, as other Agaves, is a drought tolerant desertperennial plant successfully copying with high temperatures and desiccation.Agaves are characterized by tissue succulence and Crassulacean AcidMetabolism to minimize water loss. The presence of a highly acetylated ligninin Agaves will increase hydrophobicity of the vascular tissues thus helping toreduce water loss in the plant. However, abaca, despite being highly acetylated,has not succulent leaves and is not drought tolerant, and therefore the highlyacetylated lignin might have a different role for this plant. In this case, the roleof the high extent of lignin acetylation could be more related with the loweraffinity of acetylated monolignols to form linkages producing a more noncondensedlignin enriched in -O-4 linkages. Whatever the reason for theoccurrence of acetylated lignin in plants, it seems that the mechanism for ligninacetylation confers the plant a high flexibility to produce different types oflignins with different degree of acetylation to adapt to different environmentalconditions.In conclusion, the presence of naturally acetylated lignin units, which in someplants make up to 80% of the uncondensed S lignin, has been largelyunderestimated. This has mainly been due to the analytical methodologies usedfor their isolation and structural characterization, which are not appropriate forthe analysis of native acetylated lignin. Therefore, all subsequent ligninstructural studies should take into account the occurrence of these moieties andaccordingly adapt the methodological protocols used.AcknowledgementsThis 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 I3Pfellowship; GM thanks the Spanish Ministry of Education for a FPI fellowship.Literature cited[1] Ralph, J. An unusual lignin from kenaf. J. Nat. Prod. 1996, 59, 341-342.[2] Sun, R.C.; Fang, J.M.; Goodwin, A.; Lawther, J.M., Bolton, A.J.Fractionation and characterization of ball-milled and enzyme lignin fromabaca fibre. J. Sci. Food Agric. 1999, 79, 1091-1098.[3] Sun, R.C.; Sun, X.F.; Wang, S.Q.; Zhu, W.; Wang, X.Y. Ester and etherlinkages between hydroxycinnamic acids and lignin from wheat, rice, rye,and barley straws, maize stems, and fast-growing poplar wood. Ind. Crops& Prod. 2002, 15, 179-188.123

5. Resultados y discusión[4] del Río, J.C.; Gutiérrez, A.; Rodríguez, I.M., Ibarra, D., Martínez, A.T.Composition of non-woody plant lignins and cinnamic acids by Py-GC/MS, Py/TMAH and FT-IR. J. Anal. Appl. Pyrol. 2007, 79, 39-46.[5] Ros Barceló, A. Lignification in plant cell walls. Int. Rev. Cytol. 1997, 176,87-132.[6] Boerjan, W.; Ralph, J.; Baucher, M. Lignin biosynthesis. Annu. Rev. PlantBiol. 2003, 54, 519–546[7] Ralph, J.; Lundquist, K.; Brunow, G.; Lu, F.; Kim, H.; Schatz, P. F.;Marita, J. M.; Hatfield, R. D.; Ralph, S. A.; Christensen, J. H. et al.Lignins: natural polymers from oxidative coupling of 4-hydroxyphenylpropanoids. Phytochem. Rev. 2004, 3, 29-60.[8] Sarkanen, K.V.; Chang, H. -M.; Allan, G.C. Species variation in lignins.III. Hardwood lignins. Tappi 1967, 50, 587-590.[9] Lu, F.; Ralph, J. Preliminary evidence for sinapyl acetate as a ligninmonomer in kenaf. Chem. Commun. 2002, 90–91.[10] Lu, F.; Ralph, J. Novel structures in lignins incorporating acylatedmonolignols. In Proceedings of 13 th 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 (Populustremula). J. Chem. Soc. 1955, 2347–2351.[12] Nakano, J.; Ishizu, A.; Migata, N. Studies on lignin. XXXII. Ester groupsof lignin. Tappi 1961, 44, 30–32.[13] Monties, B.; Lapierre, C. Donnés récentes sur l’hétérogénéite de la lignine(Recent data on the heterogeneity of lignin). Physiol. Veg. 1981, 19, 327-348.[14] Landucci, L. L.; Deka, G. C.; Roy, D. N. A. 13 C NMR study of milledwood 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 asrevealed by NMR. J. Am. Chem. Soc. 1994, 116, 9448-9456.[16] Sun, R. C.; Fang, J. M.; Tomkinson, J. Fractional isolation and structuralcharacterization of lignins from oil palm trunk and empty fruit bunchfibres. J. Wood Chem. Technol. 1999, 19, 335–356.[17] Lu, F. and Ralph, J. Detection and determination of p-coumaraloylatedunits in lignin. J. Agric. Food Chem. 1999, 47, 1985-1992.124

5. Resultados y discusión[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 inpoplar xylem upon down-regulation of caffeoyl-coenzyme A O-methyltransferase, an enzyme involved in lignin biosynthesis. J. Biol.Chem. 2000, 275, 36899–36909.[19] del Río, J. C.; Gutiérrez, A.; Martínez A. T. Identifying acetylated ligninunits in non-wood fibers using pyrolysis-gas chromatography/massspectrometry. Rapid Commun. Mass Spectrom. 2004, 18, 1181-1185.[20] Gutiérrez, A.; Rodríguez, I. M.; del Río J. C. Chemical characterization oflignin and lipid fractions in kenaf bast fibers used for manufacturing highqualitypapers. J. Agric. Food Chem. 2004, 52, 4764-4773.[21] del Río, J. C.; Gutiérrez, 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 (DFRCmethod), a new method for lignin analysis: protocol for analysis of DFRCmonomers. J. Agric. Food Chem. 1997, 45, 2590-2592.[23] Lu, F.; Ralph, J. The DFRC method for lignin analysis. Part 1. A newmethod for aryl ether cleavage: lignin model studies. J. Agric. FoodChem. 1997, 45, 4655-4660.[24] Lu, F.; Ralph, J. The DFRC method for lignin analysis. 2. Monomers fromisolated lignin. J. Agric. Food Chem. 1998, 46, 547-552.[25] Ralph, J.; Lu, F. The DFRC method for lignin analysis. 6. A simplemodification for identifying natural acetates in lignin. J. Agric. Food Chem.1998, 46, 4616-4619.[26] Björkman, A. Studies on finely divided wood. Part I. Extraction of ligninwith neutral solvents. Sven. Papperstidn. 1956, 59, 477-485.[27] Sederoff, R. D.; MacKay, J.; Ralph, J.; Hatfield, R. Unexpected variation inlignin. Curr. Opin. Plant Biol. 1999; 2: 145.[28] Takahama, U.; Oniki, T.; Shimokawa, H. A possible mechanism for theoxidation of sinapyl alcohol by peroxidase-dependent reactions in theapoplast: enhancement of the oxidation by hydroxycinnamic acids andcomponents of the apoplast. Plant Cell Physiol. 1996, 37, 499-504.[29] Ralph, J. Elucidation of new pathways in normal and perturbedlignification. Appita 2005, 3-13.125

5. Resultados y discusiónPublicación III:del Río J.C., Rencoret J., Marques G., Gutiérrez A., Ibarra D., Santos J.I.,Jiménez-Barbero J., Zhang L. and Martínez A.T. (2008) Highly acylated(acetylated and/or p-coumaroylated) native lignins from diverse herbaceousplants. Journal of Agricultural and Food Chemistry, 56, 9525-9534.126

5. Resultados y discusiónHighly acylated (acetylated and/or p-coumaroylated) native lignins fromdiverse herbaceous plantsJosé C. del Río † , Jorge Rencoret † , Gisela Marques † , Ana Gutiérrez † , David Ibarra ‡ , J. IgnacioSantos ‡ , Jesús Jiménez-Barbero ‡ , Liming Zhang ¥ , Ángel T. Martínez ‡† Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, P.O. Box 1052, 41080-Seville, Spain‡ Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain¥ Royal Institute of Technology (KTH), Fiber and Polymer Technology, SE-100 44Stockholm, SwedenAbstractThe structure of lignins isolated from the herbaceous plants sisal (Agavesisalana), kenaf (Hibiscus cannabinus), abaca (Musa textilis) and curaua(Ananas erectifolius) has been studied upon spectroscopic (2D-NMR) andchemical degradative (Derivatization Followed by Reductive Cleavage)methods. The analyses demonstrate that the structure of the lignins from theseplants is highly remarkable, being extensively acylated at the -carbon of thelignin 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 curauaare esterified with acetate and p-coumarate groups. The structures of all thesehighly-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 astrikingly low proportion of traditional -´linkages, which indeed arecompletely absent in the lignins from abaca and curaua. The occurrence of -´homo-coupling and cross-coupling products of sinapyl acetate in the ligninsfrom sisal and kenaf indicates that sinapyl alcohol is acetylated at monomerstage and that, therefore, sinapyl acetate should be considered as a realmonolignol involved in the lignification reactions.Keywords: lignin, herbaceous plants, sinapyl acetate, sinapyl p-coumarate, 2D-NMR, HSQC, DFRC, sisal, kenaf, abaca, curaua1. IntroductionLignins are complex natural biomacromolecules characteristics of vascularplants, where they provide mechanical support. In addition, lignin waterproofsthe cell wall, enabling transport of water and solutes through the vascularsystem, and plays a role in protecting plants against pathogens (1). The ligninpolymer results from the random oxidative coupling of p-hydroxycinnamylmonolignols mediated by laccases and/or peroxidases (2, 3). The three primary127

5. Resultados y discusiónmonolignols are p-coumaryl, coniferyl and sinapyl alcohols, which produce,respectively, p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S)phenylpropanoid units when incorporated into the lignin polymer.However, it is now widely accepted that other monomers also participate incoupling reactions giving rise to the lignin macromolecule. This is the case of -acylated (with acetate, p-hydroxybenzoate and/or p-coumarate groups) ligninswhich have been discovered in many plants. Different grass lignins are partiallyp-coumaroylated and some hardwood lignins such as poplar, aspen or willowlignins are p-hydroxybenzoylated (3-11). Acetylated lignin units have also beenreported to occur in many plants (12-16). Characteristic products from sinapyland coniferyl acetate coupling have been detected upon degradative techniques(Py-GC/MS and derivatization followed by reductive cleavage, DFRC) in thelignin of several plants characterized by having a high S/G ratio such as sisal,kenaf, abaca and jute (15, 16). Previous studies have shown that lignin fromthese plants is acetylated exclusively at the -position of the side-chain and thatthis acetylation occurred predominantly on S-units (12-16). Moreover, thesestudies have provided strong evidence that sinapyl acetate is implicated as amonomer in lignification in several plants and that the naturally acetylated ligninderives not from acetylation of the lignin polymer but from polymerization ofpre-acetylated monolignols (14, 16, 17). The same seems also to occur withsinapyl p-coumarate and sinapyl p-hydroxybenzoate (17-19).We have recently shown, using a previously developed modification of theDFRC method to allow detection of natural acetate groups (13) (the so-calledDFCR´ methodology) that lignin -acetylation is widespread, and probablyubiquitous, among angiosperms, although at different extents, but is absent fromconifers (16). Moreover, the lignins of many plants (e.g. the non-woody sisal,kenaf, abaca or the hardwood hornbeam) are particularly extensively acetylated(up to 80% of the S-lignin moieties) (16). However, the DFRC degradationmethod only cleaves - and -aryl ether bonds allowing only the analysis of themonomeric degradation products released from non-condensed etherified ligninunits. Therefore, the DFRC method, although extremely useful, does not giveinformation of the whole macromolecule, and the extent of lignin acylation maybe actually different from that estimated.Spectroscopic techniques, and particularly 1D- and 2D-NMR, can provideinformation of the structure of the whole macromolecule and are powerful toolsfor lignin structural elucidation (7, 20-24). Therefore, they can be very useful toestimate the actual extent of lignin acylation. The main advantage ofspectroscopic techniques over degradation methods is the analysis of the wholelignin structure and direct determination of the different lignin moieties andinter-unit linkages. In this paper, we study the structure of some naturallyextensively acylated lignins occurring in several herbaceous plants (namelysisal, kenaf, abaca and curaua) by a combination of chemical degradative(DFRC) and spectroscopic (2D-NMR) techniques.128

5. Resultados y discusión2. Material and methods2.1. SamplesThe plant samples selected for this study consist of bast fibers obtained from thestalk phloem layer of kenaf (Hibiscus cannabinus) and leaf fibers of sisal (Agavesisalana), abaca (Musa textilis) and curaua (Ananas erectifolius). The fiberswere finely ground to sawdust using a knife mill (Analysenmühle A10, Jankeand Kunkel GmbH, Staufen, Germany) before analysis. Milled-wood lignin(MWL) was extracted from finely ball-milled (150 h) plant material, free ofextractives and hot water soluble material, using dioxane-water (9:1, v/v),followed by evaporation of the solvent, and purified as described (25). The finalyields ranged from 5-15% of the original lignin content. Extension of millingtime, which would increase yield, was avoided to prevent chemicalmodifications on the lignin structure.2.2. DFRC (derivatization followed by reductive cleavage)The DFRC degradation was performed according to the developed protocol (26-28). Lignins (10 mg) were stirred for two hours at 50ºC with acetyl bromide inacetic acid (8:92). The solvents and excess of bromide were removed by rotaryevaporation. The products were then dissolved in dioxane/acetic acid/water(5:4:1, v/v/v), and 50 mg powered Zn was added. After 40 min stirring at roomtemperature, the mixture was transferred into a separatory funnel withdichloromethane and saturated ammonium chloride. The pH of the aqueousphase was adjusted to less than 3 by adding 3% HCl, the mixture vigorouslymixed and the organic layer separated. The water phase was extracted twicemore with dichloromethane. The combined dichloromethane fractions weredried over anhydrous NaSO 4 and the filtrate was evaporated in a rotaryevaporator. The residue was acetylated for 1 h in 1.1 mL of dichloromethanecontaining 0.2 mL of acetic anhydride and 0.2 mL pyridine. The acetylatedlignin degradation products were collected after rotary evaporation of thesolvents, and subsequently analyzed by GC/MS. To asses the presence ofnaturally acetylated lignin units, a modification of the standard DFRC methodby using propionylating instead of acetylating reagents (DFRC´) was used in thepresent study (13, 16).The GC/MS analyses were performed with a Varian model Star 3800 GCequipped with an ion trap detector (Varian model Saturn 4000) using a mediumlength(15 m) capillary column (DB-5HT, 5 m × 0.25 mm I.D., 0.1 m filmthickness) from J&W Scientific. The oven was heated from 120 (1 min) to 330ºC at 6 ºC/min, and held for 4 min at the final temperature. The injector wasprogrammed from 60ºC to 350ºC at a rate of 200ºC/min and held until the end ofthe analysis. The transfer line was kept at 300 ºC. Helium was used as the carriergas at a rate of 2 mL/min. Quantification of the released individual monomerswas performed using tetracosane as external standard, and by assuming similar129

5. Resultados y discusiónresponse factors as those of the acetylated monomers previously reported (26),although without authentication on our instrument. Molar yields were calculatedon the basis of molecular weights of the respective acetylated and/orpropionylated compounds.2.3. NMR spectroscopyNMR spectra were recorded at 25 ºC on a Bruker AVANCE 500 MHz equippedwith a z-gradient triple resonance probe. Around 40 mg of lignin were dissolvedin 0.75 mL of deuterated dimethylsulfoxide (DMSO-d 6 ) and 2D-NMR spectrawere recorded in HSQC (heteronuclear single quantum correlation) experiments.The spectral widths were 5000 Hz and 13200 Hz for the 1 H- and 13 C-dimensions, respectively. The number of collected complex points was 2048 for1 H-dimension with a recycle delay of 5 s. The number of transients was 64, and256 time increments were always recorded in 13 C-dimension. The 1 J CH used was140 Hz. The J-coupling evolution delay was set to 3.2 ms. Squared cosine-bellapodization function was applied in both dimensions. Prior to Fourier transformthe data matrixes were zero filled up to 1024 points in the 13 C-dimension. Thecentral solvent peak was used as an internal reference ( C 40.1; H 2.50 ppm).HSQC cross-signals were assigned by combining the results of the differentexperiments, and comparing with the literature (20-23, 29-32). Asemiquantitative analysis of the intensities of the HSQC cross-signal intensitieswas performed (29, 33). Since the cross-signal intensity depends on theparticular 1 J CH value, as well on the T 2 relaxation time, a direct analysis of theintensities is impossible. A more accurate quantification of the 2D HSQC NMRdata analysis was achieved by using the recently published method in which theerrors caused by T 2 relaxation, off-resonance effect and coupling constantdeviation could mostly be eliminated (34). Thus, the integration on the crosssignalswas performed separately for the different regions of the HSQC spectra,which contain signals that correspond to chemically analogous carbon-protonpairs. For these signals, the 1 J CH coupling value is relatively similar, theirchemical shifts are also similar to each other hence the error of off-resonanceeffect is small, and therefore can be used semiquantitatively to estimate therelative abundance of the different species. In the aliphatic oxygenated region,inter-unit linkages were estimated from C -H correlations to avoid possibleinterference from homonuclear 1 H- 1 H couplings, and the relative abundance ofside-chains involved in inter-unit linkages were calculated. In the aromaticregion, C 2,6 -H 2,6 correlations from S units and C 2 -H 2 plus C 6 -H 6 correlationsfrom G units were used to estimate the S/G ratio of lignin, and p-coumaric acidcontent was estimated from its C 2,6 -H 2,6 correlation signal. Lignin acylation wasestimated from the intensities of C -H correlations in acylated and non-acylatedside-chains.130

5. Resultados y discusión3. Results and discussionMWL is a lignin preparation considered as the most representative of the wholenative lignin in the plant (25), in spite of its low yield and the possibility ofsome modifications during milling (35). Therefore, in this work, the MWLisolated from the selected herbaceous plants (sisal, kenaf, abaca and curaua)were analyzed by 2D-NMR and DFRC to get insight into their structuralcharacteristics. However, we must still keep in mind that the results obtainedhere reflect only the structure of isolated MWL, which only represents a part ofthe whole lignin in the plant.3.1. HSQC-NMR spectra of highly acetylated ligninsThe HSQC NMR spectra of the different MWL showed three regionscorresponding to aliphatic, side-chain and aromatic 13 C- 1 H correlations. Thealiphatic (non-oxygenated) region showed signals with no structural information(except for the presence of acetate signals at C / H 20.7/1.74 ppm) and thereforeis not discussed in detail. The side-chain regions ( C / H 50-90/2.5-5.5 ppm) andthe aromatic regions ( C / H 95-150/5.5-8.5 ppm) of the MWL selected for thisstudy are shown in Figures 1 and 2, respectively, and the main substructuresfound in these lignins are depicted in Figure 3. The main lignin cross-signalsassigned in the HSQC spectra are listed in Table 1.In the side-chain region, cross-signals of methoxyls ( C / H 56.2/3.73 ppm)and side-chains in O-4´ substructures were the most prominent in all lignins.Interestingly, the spectra clearly show the presence of intense signalscorresponding to acylated -carbon in the range from C / H 63.5/3.83 and 4.30ppm in all lignin samples, together with the presence of signals from normalhydroxylated -carbon (at C / H 60.2/3.30 and 3.70 ppm). The HSQC spectraindicate that these lignins are extensively acylated and that acylation occursexclusively at the -position of the lignin side-chain. HSQC can not identify thenature of the ester, although it was shown in previous studies that acetates (andp-coumarates) occurred in these lignins (12, 13, 15, 16, 36). Traces (less than0.5% of acylated -carbon) of a signal at C / H 5.87/74.66 ppm corresponding toacylated -carbon were found in the HSQC of sisal and kenaf MWL although itwas absent in the rest of the lignins studied here. This signal could be due to amigration of the acetyl group from the -carbon to the -carbon in the ligninside-chain, as already advanced by Ralph (12).An estimation of the percentage of -acylation of the lignin side-chain wascalculated from the HSQC spectra by integration of the signals corresponding tothe hydroxylated and acylated -carbon (Table 2), and ranged from 58% inkenaf bast lignin up to 80% in abaca lignin. Although kenaf lignin has alreadybeen known for long to be highly acetylated (12), up to our knowledge, this isthe first time that this type of highly acylated lignin has been described in other131

5. Resultados y discusiónplants. The high extent of lignin acylation observed in different herbaceousplants (including both mono- and eudicotyledoneous) indicates that this type oflignin might be more frequent than previously thought. Naturally acetylatedlignins may also occur in many other plants but their occurrence has probablybeing biased due to the limitations of the analytical procedures used for theirisolation and/or structural characterization. Natural acetates present on ligninmight have been hydrolyzed and removed when using traditional isolationmethods (such as alkaline extraction often applied to non-wood lignins) anddegradative procedures for chemical characterization (such as nitrobenzeneoxidation, CuO oxidation or thioacidolysis). Indeed, for spectroscopic analysis,e.g. using NMR, lignin is frequently in vitro acetylated for improved solubilityand spectroscopic properties, which prevented the detection of natural ligninacetylation.(a)MeOA /A´D ´ C C A carbohydrates70758085D A' A' C DA 556065 H 5.0 4.5 4.0 3.5 3.0 C(b)60MeO C D 55A' A H 5.0 4.5 4.0 3.5 3.0A /A´ C 657075A' carbohydrates 80A 85 C(c)A /A´ /A´´ HMeOA' /A'' D A D D ´ AcarbohydratesA' /A'' 5.0 4.5 4.0 3.5 3.0 C55606570758085D D ´ C C(d)A' /A'' A /A´ /A´´D A' /A'' D ´ A H5.0 4.5 4.0MeOA carbohydrates3.5 3.055606570758085Figure 1. Expanded side-chain region, C / H 50-90/2.5-5.5 ppm, of the HSQC spectra of thelignins from (a) sisal, (b) kenaf, (c) abaca and (d) curaua. Carbohydrate signals are presentedin grey color. See Table 1 for signal assignment and Figure 3 for the main lignin structures(A-D) identified.132

5. Resultados y discusión(a)S 2,6(b)100S 2,6 (C =O)S/G 3.9 S/G 5.6 CS 2,6 (C =O)S 2,6 C100D 2`110D 2`110G 2G 5G 6G 2G 5G 6D 6`120D 6`120130130140140 H8.07.57.06.56.0 H8.07.57.06.56.0(c)S 2,6100(d)S/G 8.7S 2,6 (C =O)S/G 4.9D 2` 110G 2 A'' ´G 5G 6D 6` 120A'' ´140A'' 2´,6´A'' 3´,5´130 CS 2,6 (C =O)A'' 2´,6´G 2G 5G 6S 2,6D 2`D 6`A'' ´A'' 3´,5´A'' ´ C100110120130140 H8.07.57.06.56.0Figure 2. Expanded aromatic region, C / H 95-150/5.5-8.5 ppm, of the HSQC spectra of thelignins from (a) sisal, (b) kenaf, (c) abaca and (d) curaua. See Table 1 for signal assignmentand Figure 3 for the main lignin structures (A-D) identified. G and S are the guaiacyl andsyringyl aromatic units, respectively. H8.07.57.06.56.0The side-chain region of the HSQC spectra gives also additional informationabout the inter-unit linkages present in the structure of these lignins. All thespectra showed prominent signals corresponding to O-4´ aryl ether linkages.The C -H correlations in O-4´ substructures were observed at C / H72.3/4.86 ppm (structures A, A' and A''), while the C -H correlations wereobserved at C / H 86.5/4.10 ppm in normal -OH O-4´ aryl ethersubstructures (A) but shifted to C / H 83.6/4.32 ppm in -acylated O-4´ arylether substructures (A', A''). O-4´ aryl ether substructures were highlypredominant in all the lignins analyzed here although other substructures werealso observed. Small signals corresponding to spirodienone (1´, O´linkages) substructures (D) can be observed in the spectra of sisal, kenaf, abacaand curaua lignins. Signals of spirodienone C -H , C ´-H ´ and C -H correlations were observed at C / H 85.4/4.64, 85.4/4.80 and 56.1/3.09 ppm,respectively. Spirodienone substructures were previously reported in the lignin133

5. Resultados y discusiónTable 1. Assignment of Main Lignin 13 C- 1 H Cross-Signals in the MWL HSQC SpectraShown in Figures 1 and 2. C / H (ppm)Assignment53.7/3.12 C -H in ' (resinol) substructures (C)56.1/3.09 C -H in '(spirodienone) substructures (D)60.0/3.38-3.71 C H in -O-4' substructures (A)63.8/3.83-4.30 C H in -acylated -O-4' substructures (A' and A'')71.7/3.81 and 4.17 C -H in ' (resinol) substructures (C)72.3/4.86 C -H in -O-4' substructures (A, A' and A'')82.1/5.12 C -H in '(spirodienone) substructures (D)83.6/4.32 C -H in -acylated -O-4' substructures (A' and A'')85.4/4.64 C -H in ' (resinol) substructures (C)85.4/4.80 C ´-H ´ in '(spirodienone) substructures (D)86.5/4.10 C -H in -OH -O-4' substructures (A)87.7/5.45 C -H in phenylcoumaran substructures (B)103.8/6.68 C 2 -H 2 and C 6 -H 6 in syringyl units106.7/7.36 and 7.21 C 2 -H 2 and C 6 -H 6 in oxidized (C =O) syringyl units111.5/6.99 C 2 -H 2 in guaiacyl units111.6/6.23 C 2´-H 2´ in '(spirodienone) substructures (D)114.3/6.24 C ´-H ´ in p-coumaroylated substructures (A'')115.2/6.71 and 6.94 C 5 -H 5 in guaiacyl units116.2/6.77 C 3´-H 3´ and C 5´-H 5´ in p-coumaroylated substructures (A'')118.3/6.19 C 6´-H 6´ in (spirodienone) substructures (D)119.5/6.83 C 6 -H 6 in guaiacyl units130.5/7.4 C 2´-H 2´ and C 6´-H 6´ in p-coumaroylated substructures (A'')145.1/7.39 C ´-H ´ in p-coumaroylated substructures (A'')from kenaf bast fibers by Zhang et al. (37). Phenylcoumaran (5´ linkages)substructures (B) were also found, although in very small proportions. Veryweak signals corresponding to C -H correlations of phenylcoumaransubstructures at C / H 87.7/5.45 ppm were observed in the spectra of sisal, kenafand curaua lignins, but were absent in the spectrum of abaca. The presence ofthese low amounts of phenylcoumaran substructures was expected due to thevery low levels of guaiacyl lignin units in all these samples. Finally, resinol(´ linkages substructures (C) were clearly observed in the spectrum ofkenaf. Signals for the C -H , C -H and the double C -H correlations of resinolsubstructures were observed at C / H 85.4/4.64, 53.7/3.12 and 71.7/3.81 and4.17 ppm, respectively. Resinol substructures could also be observed, althoughin very small traces, in the spectrum of sisal, but were completely absent in thespectra of abaca and curaua lignins. The relative abundances of the main interunitlinkages present in the MWL selected for this study were calculated fromthe HSQC spectra and are shown in Table 2. All these highly acetylated ligninsshare a common characteristic, the strikingly high proportion of O-4´ etherlinkages (up to 94% of all linkages) and a very low proportion of condensedlinkages (i.e. ´, ´ and ´). Some of these condensed linkages (´and ´) are even absent in some lignins (abaca and curaua).134

5. Resultados y discusiónTable 2. Structural characteristics (percentage of -acylation, relative abundance of the maininter-unit linkages, and S/G ratio) observed from the HSQC spectra of the selected MWL.sisal kenaf abaca curauaPercentage of -acylation 68 58 80 69Linkage relative abundance (% of side-chains involved)-O-4' alkyl-aryl ether 89 84 94 94-1' (spirodienone) 5 6 6 4-5' (phenylcoumaran) 2 2 0 2-' (syringaresinol) 4 8 0 0S/G ratio 3.9 5.6 8.7 4.9The main cross-signals in the aromatic region of the HSQC spectra (Figure 2)correspond to the aromatic rings of the different lignin units. Signals fromsyringyl- (S) and guaiacyl- (G) lignin units can be observed in all spectra. Thesyringyl units show a prominent signal for the C 2,6 -H 2,6 correlation at C / H103.8/6.68 ppm, while guaiacyl units showed different correlations for C 2 -H 2( C / H 111.5/6.99 ppm), C 5 -H 5 ( C / H 115.2/6.71 and 6.94) and C 6 -H 6 ( C / H119.5/6.83 ppm). Signals corresponding to C 2,6 -H 2,6 correlations in C-oxidizedS-lignin units were observed at C / H 106.7/7.36 and 7.21 ppm. No signals for p-hydroxyphenyl (H) lignin units could be detected in the HSQC spectra of theselignins. An estimation of the relative proportions of the S and G-lignin units inthe HSQC spectra revealed that all the lignins selected for this study present avery high S/G ratio, ranging from 3.9 in sisal to 8.7 in abaca (Table 2). Othersignals present in this region of the HSQC spectra are from spirodienonesubstructures (D) with C 2´-H 2´ and C 6´-H 6´ correlations at C / H 111.6/6.23 and118.3/6.19, respectively. Prominent signals corresponding to p-coumaratestructures were observed in the lignins of abaca and curaua. Cross-signalscorresponding to the correlations C 2´,6´-H 2´,6´ at C / H 130.5/7.40 ppm and C 3´,5´-H 3´,5´ at C / H 116.2/6.77 ppm of the aromatic ring and signals for thecorrelations of the unsaturated C ´-H ´ at C / H 145.1/7.39 and C ´-H ´ at114.3/6.24 ppm of the p-coumarate unit in structure A'' of Figure 3, wereobserved in this region of the HSQC spectra of abaca and curaua. In abacalignin, p-coumaric acid has already been reported to be esterified to the ligninpolymer (8, 16, 35, 38).3.2. Degradation Followed by Reductive Cleavage (DFRC and DFRC´)The HSQC data shown above indicate that these lignins are extensively acylatedat the -position of the side-chain, but cannot provide additional information on135

5. Resultados y discusiónthe nature of the acyl group (besides the occurrence of acetate and p-coumaratemoieties). A sensitive and selective method is therefore needed to reveal theHO4´5´6´O3´2´1´´´´OHOHOMeOO5’4’6’1’2’3’HOOMeOO5’4’6’1’2’3’HOOMeOO5'’4'’6'’1'’2'’3'’612OMe612OMe612OMeMeO543OMeMeO543OMeMeO543OMeOOOAA'A''HOMeO651’6’5’4’O1234O2’3’OMeOMeMeOO546312OO’’’2’1’OMe3’4’5’6’OOMeMeOOHOOMe432561 MeOO ´MeO1´6´ 2´5´ 3´4´6''5''1''2''3''4''O´´OMeOHOMeOMeOBCFigure 3. Main structures present in the highly acylated lignins studied here: (A) -4´ arylether linkages; (A') -4´ aryl ether linkages with acetylated -carbon; (A'') -4´ arylether linkages with p-coumaroylated -carbon; (B) phenylcoumaran structures formed by5´ and -4´ linkages; (C) resinol structures formed by ´, O´and O´linkages; and (D) spirodienone structures formed by 1´,O-´ linkages.Dnature of the acyl group that is esterifying the -carbon of the lignin side-chainand to know to which lignin moiety it is attached. The DFRC degradationmethod, which cleaves - and -ether linkages in the lignin polymer leaving -esters intact (26-28), seems to be the most appropriate method for the analysis ofnative -acylated lignin.DFRC analysis of the lignin samples selected for this study allowedconfirming that p-coumarate groups are attached to the -carbon of abaca andcuraua lignins, and predominantly on syringyl units (Figure 4). Saturated p-coumarate (dihydro-p-coumarate) esterified to sinapyl alcohol (as its acetatederivative, S dpc ) was expected to be the major DFRC degradative compound,136

5. Resultados y discusiónaccording to Lu and Ralph (9), and this was the only degradation product thatwas quantified in our previous paper (16). However, a closer look to other majordegradation compounds produced upon DFRC of abaca and curaua ligninsindicated the release of important amounts of the unsaturated counterpart, thatis, intact sinapyl p-coumarate (as its acetate derivative, S pc ), that was biased, andtherefore not quantified, in our previous paper. Therefore, in this work, we havenow taken into account both compounds to quantify the total abundance ofsinapyl p-coumarate units present in these lignins (Table 3). Trace amounts ofthe respective coniferyl p-coumarate could also de detected in abaca and curaualignins. Moreover, some amounts of free p-coumaric acid (as its acetatederivative) could be observed among the DFRC degradation products of curaualignin as a broad peak (Figure 4), that could probably correspond to p-coumaricacid moieties linked to lignin through O-4´ aryl-ether bonds.The original DFRC degradation method, however, does not allow the analysisof native acetylated lignin because the degradation products are acetylatedduring the degradation procedure, but with appropriate modification of theprotocol by substituting acetylating reagents with propionylating reagents,DFRC´, it is also possible to obtain information about the occurrence of nativelignin acetylation (13). Figure 5 shows the chromatograms of the DFRC´products released from the lignin samples selected in this study. All the analyzedlignins 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 fromnormal -OH units in lignin. In addition, the presence of originally -acetylatedguaiacyl (c-G ac and t-G ac ) and syringyl (c-S ac and t-S ac ) lignin units could also beclearly observed in the chromatograms of all of the selected lignins indicatingthat acetylation occurred exclusively at the -carbon of the lignin side-chain, asalready observed in the HSQC spectra.Table 3. Abundance (Molar Yields) of the DFRC and DFRC´ degradation monomers of theMWL isolated from the different plants selected for this study, and relative abundances of thedifferent acylated (acetylated and p-coumaroylated) lignin moieties.Monomers (mol/g lignin)G G ac G pc S S ac S pca%S acb%S pcc%G acd%G pc S/Gsisal 122 124 0 108 378 0 78 0 50 0 2.0kenaf 390 38 0 543 780 0 59 0 9 0 3.1abaca 50 3 1 21 131 124 48 45 6 2 5.1curaua 250 252 3 515 595 195 46 15 50 1 2.6a %S ac is the percentage of acetylated S units respect to the total S units. b %S pc is the percentage of p-coumaroylated S units respect to the total S units. c %G ac is the percentage of acetylated G units respect to thetotal G units. d %G pc is the percentage of p-coumaroylated G units respect to the total G units137

5. Resultados y discusión100tS(a)relative intensityS pctGcSS pcS pc Sdpc0100tS(b)relative intensitytGS pcpCcSS pcS dpc S pc05 10 15 20 25Retention 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. S dpcand S pc 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 transguaiacyland syringyl monomers, respectively, (as their acetyl derivatives).The results from the DFRC and DFRC´ analysis of the MWL selected for thisstudy, namely the molar yields of the released monomers, the percentages ofnaturally acetylated guaiacyl (%G ac ) and syringyl (%S ac ) and p-coumaroylatedguaiacyl (%G pc ) and syringyl (%S pc ) lignin moieties, and the S/G ratios, arepresented in Table 3. The data indicate that a high extent of -acetylation occurs138

5. Resultados y discusiónin all lignins studied here, and that p-coumaric acid is also found partiallyesterifying the lignin of abaca and curaua, in agreement with the NMR data. Inall cases, acetate and p-coumarate groups are preferentially attached to syringylunits, as previously noted for other lignins (7-9, 12-16, 39). Interestingly, thehigh extent of acetylation observed in sisal and curaua also included the G-ligninunits (around 50% of acetylation in both cases). By contrast, in kenaf and abacalignins, the -carbon of G-lignin units is mostly not esterified. On the otherhand, the high extent of acylation of the lignin monomers observed by theDFCR (and DFRC´) method, which can only analyze non-condensed ligninmoieties, is in accordance with the high extent of lignin acylation observed byHSQC technique, which allows the analysis of the entire MWL structure,including both condensed and non-condensed linkages. This fact indicates thatboth condensed and non-condensed moieties have similar extent of acylation.However, we must now convey again that the results presented here reflect onlythe structure of the isolated MWL, which only represent a small part of theentire native lignin. However, similar lignin S/G ratio and acylation degree havebeen 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 beconsidered as the most representative preparation for the plant native lignin, inspite of its low yield.Previous papers describing the structure of some of these lignins have failedto detect their high levels of acetylation. A recent paper describing the structureof sisal lignin (41) did not detect the high levels of acetylation, despite of usingspectroscopic techniques. Probably, this was due to the method used forisolation (acidolysis) that might have hydrolyzed the acetyl groups, or to amisassignment of the spectral bands. Previous structural studies on abaca lignin(8, 16, 36, 38), using different degradation methods, also suggested theoccurrence of p-coumaroylated units attached to the -carbon of the lignin sidechain.The presence of acetylated -carbons was also observed in abaca fibersdirectly 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 frompolymerization of acylated monolignols or from acylation of the lignin polymerhas recently been addressed and sinapyl acetate has been demonstrated tobehave 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 –´ resinolstructures can not occur because the absence of free -hydroxyls needed to rearomatizethe quinone methide moiety. Instead, new tetrahydrofuran structuresare formed from the ´ homo- and cross-coupling of two sinapyl (acylatedand non-acylated) monolignols, as advanced by Lu and Ralph (14) (Figure 6). Itis clear that tetrahydrofuran structures I and II can only be formed if sinapylalcohol is pre-acetylated (at monomer stage) and then undergoes ´ coupling.139

5. Resultados y discusión100t-S ac(a)relative intensity0c-Gt-G acc-S act-Gc-G act-S act-Sc-St-S100(b)relative intensity0c-G t-G acc-S act-Gc-S100(c)relative intensity relative intensity0100c-G act-S act-Sc-G t-G act-G acc-Gc-G acc-S act-Gc-S act-Gc-Sc-St-S act-S(d)05.0 7.5 10.0 12.5 15.0Retention 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-guaiacyland syringyl monomers, respectively (as their propionylated derivatives). c-G ac , t-G ac , c-S acand t-S ac are the originally acetylated cis- and trans-guaiacyl and syringyl monomers,respectively (as their propionylated derivatives).140

5. Resultados y discusiónTherefore, the presence of these tetrahydrofuran substructures in the ligninpolymer would be indicative of the occurrence of pre-acylated monolignols. Inthis work, we have investigated the presence of the tetrahydrofuran structuresarising from ´ coupling of sinapyl acetate in the MWL selected for this studyby DFRC´. The expected DFRC´ degradation products of the tetrahydrofuran´ structures, as suggested by Lu and Ralph (14), are also indicated in Figure6. Figure 7 shows the reconstructed chromatograms (sum of the single ionchromatograms of the respective base peaks) of the DFRC´ degradation productsof the expected tetrahydrofuran dimers arising from the ´coupling of thesinapyl monolignols. Interestingly, compounds derived from the DFRC´ ofhomo-coupling (I´) and cross-coupling (II´a and II´b) of sinapyl acetate wereclearly observed in the lignins of sisal and kenaf, clearly indicating that in theselignins sinapyl alcohol is preacetylated and behaves as a real monolignolparticipating in post-coupling reactions. However, in the case of abaca andcuraua lignins, no traces of any type of ´ linkage (including syringaresinoland the new tetrahydrofuran structures) could be detected after DFRC, inagreement with the absence of these linkages observed in the HSQC spectra.The presence of cross-coupling structures of sinapyl alcohol and sinapylacetate indicates that both monolignols are produced simultaneously by theplant. Moreover, the relative abundance of the compounds released in Figure 7gives some additional information. In sisal, the relative molar abundance of theacetylated versus the non-acetylated sinapyl alcohols forming ´ linkages(taking into account that dimer I´ consists of two sinapyl acetates, dimers II´consist of one sinapyl acetate and one sinapyl alcohol, and dimer III´ consists oftwo sinapyl alcohols) is 44:56, with a slight predominance of the non-acetylatedsinapyl alcohol, whereas their relative molar abundances in ether-linkedstructures is 78:22, with a strong predominance of sinapyl acetate. A similartrend is also observed in kenaf lignin, where the relative molar abundance of theacetylated versus the non-acetylated sinapyl alcohols forming ´ linkagesis23:77, with a predominance of the non-acetylated sinapyl alcohol, whereastheir relative molar abundances in ether-linked structures is 59:41, with a strongpredominance of sinapyl acetate. This indicates that sinapyl acetate has a lowertendency to form ´ linkages than the normal sinapyl alcohol and thereforethose lignins having a high extent of acetylation would produce lower amountsof ´ linkages, as already advanced (16). This means that, probably, the highlevel of lignin acetylation is related in some way with the low presence of ´linkages. This is in agreement with the high proportions of -O-4´ aryl ethersubstructures and the low proportion of ´ substructures present in theselignins, as observed in the HSQC spectra shown above. Therefore, it seems thatthe high extent of -acetylation would favor the formation of a predominantly -O-4 lignin structure, which is indeed devoid of -´ linkages.141

5. Resultados y discusiónOOOCH 3 OOO-C-CH 3=O-C-CH 3=O-C-CH 3=O-C-CH 3OCH 3 -C-O==O-C-CH 3OPrO =DFRC´´homo-couplingCH 3 OCH 3 OO+CH 3 OOCH 3OCH 3OHOCH 3HOOCH 3OCH 3OHCH 3 OOCH 3OHCH 3 OIOPrI´; M + =616; [M-56] + = 560OCH 3 OOHCH 3 OOCH 3CH 3 OOO-C-CH 3=O-C-CH 3=OPrO-C-CH 3OOHOPrPrO=PrODFRC´´cross-coupling+CH 3 O+OHO CH 3 OOCH 3 -C-O=CH 3 OOCH 3CH 3 OOCH 3OCH 3OHOCH 3OHCH 3 OOCH 3OHCH 3 OOPrOPrII´b; M + = 630: [M-56] + = 574II´a; M + = 630; [M-56] + = 574CH 3 OIIOCH 3OHCH 3 OOCH 3OOHOHOPrOPr PrODFRC´´homo-coupling+OCH 3 OCH 3 OIIIHOOCH 3OCH 3OHCH 3 OOCH 3OHCH 3 OCH 3 OOCH 3OPrIII´; M + = 644; [M-56] + = 588Figure 4Figure 6. Structures of the tetrahydrofuran dimers arising from the´ coupling of sinapyl alcohol and sinapyl acetate. I:´ coupling product oftwo sinapyl acetates; II: ´ coupling product of a sinapyl alcohol and a sinapyl acetate; III: ´coupling product (syringaresinol) of two sinapylalcohols. The aryltetralin products expected from the DFRC´ degradation of these tetrahydrofuran moieties are also shown, with indication of theirmolecular weight and base peak in their mass spectra. Adapted from Lu and Ralph (14).O142

5. Resultados y discusión100II´a,II´bI´III´(a)0100III´relative intensityII´a,II´bI´(b)0100relative intensity(c)0100(d)relative intensityrelative intensity020.0 22.5 25.0 27.5 30.0 32.5Retention 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 ´ products containing two (I´),one (II´a and II´b) and no (III´) native acetates.143

5. Resultados y discusiónIt has been reported that “in vitro” peroxidase-H 2 O 2 oxidation of equimolaramounts of sinapyl alcohol and -acylated sinapyl alcohol produced equalamounts of the expected ´ coupled and cross-coupled products (I, II and III)shown in Figure 6, in a ratio 1:2:1, suggesting that the coupling reactions wereinsensitive to the acylation of the -carbon (17, 18). However, this is not the caseof what we have seen that occur in the plants, where sinapyl acetate seems tohave lower tendency to form ´ linkages and, therefore, a high abundance ofacetylated lignin monomers will ultimately produce a lignin with very low levelsof ´ structures during lignification. Therefore, a discrepancy exists betweenwhat is observed in vitro and in the plant. Moreover, as indicated by Lu andRalph (17), from the two possible stereoisomers of the ´ homo-dimerizationproduct of sinapyl acetate (structure I in Figure 6), the isomer produced during“in vitro” coupling reactions is not the same that is present in plants. Whetherthere is a protein (or any other mechanism) in the plant controlling the ´coupling reaction needs additional investigations.3.3. Structural features of highly acylated ligninsThe lignins selected for this study share some common structural features. First,they are characterized for being extensively acylated (with either acetate or p-coumarate groups), exclusively at the carbon of the lignin side-chain, andpreferentially over syringyl units. Moreover, all these lignins present a highpredominance of syringyl over guaiacyl lignin units, a very high predominanceof O-4´ linkages and a very low proportion of ´ and 5´ linkages andother condensed bonds that make these lignins very linear and unbranched. Inparticular, sisal and kenaf lignins present a high extent of -acylation,exclusively with acetate groups, and preferentially on S-lignin moieties in thecase of kenaf lignin and over both S- and G-lignin moieties in the case of sisallignin. In both cases, O-4´ aryl ether linkages predominate although some´ (resinol) and 1´ (spirodienones) linkages are observed in lowproportions. On the other hand, the structure of abaca lignin is assembled mostlywith syringyl units with a high extent of acylation of the -carbon with bothacetate and p-coumarate groups. O-4´ linkages are also predominant in thislignin. No ´ linkages are present but some 1´ (spirodienones) linkages canbe observed in abaca lignin. Finally, the lignin of curaua has a predominance ofS-lignin units, a predominance of O-4´ linkages and a high extent of acylationat the -carbon with acetate and p-coumarate groups, acetate groups being alsoesterifying to a high extent the -carbon of G-lignin units. In general, all thesestructural features make these lignins very different from the structural modelsalready proposed for softwood (43, 44) and hardwood (45). lignins. Allsubsequent lignin structural studies, including those in plant genetics or plantbreeding projects, should take into account the possible occurrence of lignin144

5. Resultados y discusiónacylation, which in many plants takes place at very high levels, as seen above,and which have often been overlooked in the past; otherwise the conclusionsdrawn may not be representative of the real native lignin structure.4. ConclusionsThe structure of the MWL isolated from the herbaceous plants sisal, kenaf,abaca and curaua has been elucidated by 2D-NMR and DFRC techniques. Theanalyses indicated that the lignins from these plants are extensively acylated atthe -carbon of the lignin side-chain (with either acetate and/or p-coumarategroups) and preferentially on syringyl moieties. The structure of these highlyacetylated lignins can be essentially regarded as syringyl units linked mostlythrough O-4´ ether bonds, where the -carbons of the side-chains areextensively acylated. The lignin polymer is therefore extremely linear andunbranched. The study of highly acylated lignins will significantly contribute toredefine the structure of lignin and to complete the lignin biosynthetic pathway.AcknowledgementsThis study has been supported by the Spanish MEC (projects AGL2005-01748and BIO2007-28719-E) and the EU contract NMP2-CT-2006-26456(BIORENEW). JR thanks the Spanish CSIC for an I3P fellowship; GM thanksthe Spanish Ministry of Education for a FPI fellowship.References(1) Sarkanen, K. V.; Ludwig, C. H. Definition and Nomenclature. In Lignins:Occurrence, Formation, Structure, and Reactions, Sarkanen, K.V. andLudwig, C.H., Ed. Wiley- Intersci.: New York, 1971: pp 1-16.(2) Boerjan, W.; Ralph, J.; Baucher, M. Lignin biosynthesis. Annu. Rev. PlantBiol. 2003, 54, 519–546.(3) Ralph, J.; Lundquist, K.; Brunow, G.; Lu, F.; Kim, H.; Schatz, P. F.;Marita, J. M.; Hatfield, R. D.; Ralph, S. A.; Christensen, J. H.; Boerjan, W.Lignins: natural polymers from oxidative coupling of 4-hydroxyphenylpropanoids. Phytochem. Rev. 2004, 3, 29-60.(4) Smith, D. C. C. p-Hydroxybenzoate groups in the lignin of aspen (Populustremula). J. Chem. Soc. 1955, 2347-2351.(5) Nakano, J.; Ishizu, A.; Migata, N. Studies on lignin. XXXII. Ester groupsof lignin. Tappi 1961, 44, 30-32.(6) Landucci, L. L.; Deka, G. C.; Roy, D. N. A. 13 C NMR study of milledwood lignins from hybrid Salix clones. Holzforschung 1992, 46, 505-511.145

5. Resultados y discusión(7) 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 asrevealed by NMR. J. Am. Chem. Soc. 1994, 116, 9448-9456.(8) Sun, R. C.; Fang, J. M.; Goodwin, A.; Lawther, J. M.; Bolton, J.Fractionation and characterization of ball-milled and enzyme lignins fromabaca fibre. J. Sci. Food Agric. 1999, 79, 1091-1098.(9) Lu, F.; Ralph, J. Detection and determination of p-coumaraloylated units inlignin. J. Agric. Food Chem. 1999, 47, 1985-1992.(10) Meyermans, H.; Morreel, K.; Lapierre, C.; Pollet, B.; De Bruyn, A.;Busson, R.; Herdewijn, P.; Devreese, B.; Van Beeumen, J.; Marita, J. M.;Ralph, J.; Chen, C.; Burggraeve, B.; Van Montagu, M.; Messens, E.;Boerjan, W. Modification in lignin and accumulation of phenolicglucosides in poplar xylem upon down-regulation of caffeoyl-coenzyme AO-methyltransferase, an enzyme involved in lignin biosynthesis. J. Biol.Chem. 2000, 275, 36899-36909.(11) Crestini, C.; Argyropoulos, D. S. Structural analysis of wheat straw ligninby quantitative 31 P and 2D NMR spectroscopy. The occurrence of esterbonds and -O-4 substructures. J. Agric. Food Chem. 1997, 45, 1212-1219.(12) Ralph, J. An unusual lignin from kenaf. J. Nat. Prod. 1996, 59, 341-342.(13) Ralph, J.; Lu, F. The DFRC method for lignin analysis. 6. A simplemodification for identifying natural acetates in lignin. J. Agric. Food Chem.1998, 46, 4616-4619.(14) Lu, F.; Ralph, J. Preliminary evidence for sinapyl acetate as a ligninmonomer in kenaf. Chem. Commun. 2002, 90-91.(15) del Río, J. C.; Gutiérrez, A.; Martínez A. T. Identifying acetylated ligninunits in non-wood fibers using pyrolysis-gas chromatography/massspectrometry. Rapid Commun. Mass Spectrom. 2004, 18, 1181-1185.(16) del Río, J. C.; Marques, G.; Rencoret, J.; Martínez A. T.; Gutiérrez, A.Occurrence of naturally acetylated lignin units. J. Agric. Food Chem. 2007,55, 5461-5468.(17) Lu, F.; Ralph, J. Novel structures in lignins incorporating acylatedmonolignols. Appita 2005, 233-237.(18) Lu, F.; Ralph, J.; Morreel, K.; Messens, E.; Boerjan, W. Preparation andrelevance of a cross-coupling product between sinapyl alcohol and sinapylp-hydroxybenzoate. Org. Biomol. Chem. 2004, 2888-2890.146

5. Resultados y discusión(19) Morreel, K.; Ralph, J.; Kim, H.; Lu, F.; Goeminne, G.; Ralph, S. A.;Messens, E.; Boerjan, W. Profiling of oligolignols reveals monolignolscoupling conditions in lignifying poplar xylem. Plant Physiol. 2004, 136,3537-3549.(20) Ralph, J.; Marita, J. M.; Ralph, S. A.; Hatfield, R. D.; Lu, F.; Ede, R. M.;Peng, J.; Quideau, S.; Helm, R. F.; Grabber, J. H.; Kim, H.; Jimenez-Monteon, G.; Zhang, Y.; Jung, H. -J. G.; Landucci, L. L.; MacKay, J. J.;Sederoff, R. R.; Chapple, C.; Boudet, A. M. Solution-state NMR of lignin.In Advances in lignocellulosics characterization, Argyropoulos, D. S., Ed.;Tappi Press: Atlanta, 1999; pp 55-108.(21) Ralph, S. A.; Ralph, J.; Landucci, L. NMR database of lignin and cell wallmodel compounds; US Forest Prod. Lab., One Gifford Pinchot Dr.,Madison, WI 53705, 2004(http://ars.usda.gov/Services/docs.htm?docid=10491) (accessed: July2006): 2004.(22) Capanema, E. A.; Balakshin, M. Y.; Kadla, J. F. A comprehensiveapproach for quantitative lignin characterization by NMR spectroscopy. J.Agric. Food Chem. 2004, 52, 1850-1860.(23) Capanema, E. A.; Balakshin, M. Y.; Kadla, J. F. Quantitativecharacterization of a hardwood milled wood lignin by nuclear magneticresonance spectroscopy. J. Agric. Food Chem. 2005, 53, 9639-9649.(24) Balakshin, M. Y.; Capanema, E. A.; Chen, C.- L.; Gracz, H. S. Elucidationof the structures of residual and dissolved pine kraft lignins using anHMQC NMR technique. J. Agric. Food Chem. 2003, 51, 6116-6127.(25) Björkman, A. Studies on finely divided wood. Part I. Extraction of ligninwith neutral solvents. Sven. Papperstidn. 1956, 59, 477-485.(26) Lu, F.; Ralph, J. Derivatization followed by reductive cleavage (DFRCmethod), a new method for lignin analysis: protocol for analysis of DFRCmonomers. J. Agric. Food Chem. 1997, 45, 2590-2592.(27) Lu, F.; Ralph, J. The DFRC method for lignin analysis. Part 1. A newmethod for aryl ether cleavage: lignin model studies. J. Agric. FoodChem. 1997, 45, 4655-4660.(28) Lu, F.; Ralph, J. The DFRC method for lignin analysis. 2. Monomers fromisolated lignin. J. Agric. Food Chem. 1998, 46, 547-552.147

5. Resultados y discusión(29) Liitiä, T. M.; Maunu, S. L.; Hortling, B.; Toikka, M.; Kilpeläinen, I.Analysis of technical lignins by two- and three-dimensional NMRspectroscopy. J. Agric. Food Chem. 2003, 51, 2136-2143.(30) Ämmälahti, E.; Brunow, G.; Bardet, M.; Robert, D.; Kilpeläinen, I.Identification of side-chain structures in a poplar lignin using threedimensionalHMQC-HOHAHA NMR spectroscopy. J. Agric. Food Chem.1998, 46, 5113-5117.(31) Ibarra, D.; Chávez, M. I.; Rencoret, J.; del Río, J. C.; Gutiérrez, A.;Romero, J.; Camarero, S.; Martínez, M. J.; Jiménez-Barbero, J.; Martínez,A. T. Lignin modification during Eucalyptus globulus kraft pulpingfollowed by totally chlorine free bleaching: a two dimensional nuclearmagnetic resonance, Fourier transform infrared, and pyrolysis-gaschromatography/mass spectrometry study. J. Agric. Food Chem. 2007, 55,3477-3490.(32) Ibarra, D.; Chávez, M. I.; Rencoret, J.; del Río, J. C.; Gutiérrez, A.;Romero, J.; Camarero, S.; Martínez, M. J.; Jiménez-Barbero, J.; MartínezA. T. Structural modification of eucalypt pulp lignin in a totally chlorinefree bleaching sequence including a laccase-mediator stage. Holzforschung2007, 61, 634-646.(33) Heikkinen, S.; Toikka, M. M.; Karhunen, P. T.; Kilpeläinen, I. A.Quantitative 2D HSQC (Q-HSQC) via suppression of J-dependence ofpolarization transfer in NMR spectroscopy: Application to wood lignin. J.Am. Chem. Soc. 2003, 125, 4362-4367.(34) Zhang, L.; Gellerstedt, G. Quantitative 2D HSQC NMR determination ofpolymer structures by selecting the suitable internal standard references.Magn. Reson. Chem., 2007, 45(1), 37-45.(35) Holtman, K. H.; Chang, H. M.; Jameel, H.; Kaddla, J. F. Quantitative 13 CNMR characterization of milled wood lignins isolated by different millingtechniques. J. Wood Chem. Technol. 2006, 26, 21-34.(36) del Río, J. C.; Gutiérrez, A.; Rodríguez, I. M.; Ibarra, D.; Martínez, A. T.Composition of non-woody plant lignins and cinnamic acids by Py-GC/MS, Py/TMAH and FT-IR. J. Anal. Appl. Pyrolysis 2007, 79, 39-46.(37) Zhang, L; Gellerstedt, G.; Ralph, J.; Lu, F. NMR studies on the occurrenceof spirodienone structures in lignins. J. Wood Chem. Technol. 2006, 26, 65-79.148

5. Resultados y discusión(38) del Río, J. C.; Gutiérrez, 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.(39) Grabber, J. H.; Quideau, S.; Ralph, J., p-Coumaroylated syringyl units inmaize lignin; implications for -ether cleavage by thioacidolysis.Phytochemistry 1996, 43, 1189-1194.(40) Rencoret, J.; Marques, G.; Gutiérrez, A.; Nieto, L.; Santos, J. I.; Jiménez-Barbero, J.; Martínez, A. T.; del Río, J. C. “In situ” analysis of lignin by2D-NMR of wood (Eucalyptus globulus and Picea abies) and non-woody(Agave sisalana) plant materials at the gel state. Proc. EWLP-2008,Stockholm, 26-29 August.(41) Megiatto, J. D.; Hoareau, W.; Gardrat, C.; Frollini, E.; Castellain, A. Sisalfibers: surface chemical modification using reagent obtained from arenewable source; characterization of hemicellulose and lignin as modelstudy. J. Agric. Food Chem. 2007, 55, 8576-8584.(42) Ralph, J. What makes a good monolignol substitute? In The Science andLore of the Plant Cell Wall Biosynthesis, Structure and Function, Hayashi,T., Ed. Universal Publishers (BrownWalker Press): Boca Raton, FL, 2006;pp 285-293.(43) Adler, E. Lignin chemistry – past, present and future. Wood Sci. Technol.1977, 11, 169-218.(44) Brunow, G. Methods to Reveal the Structure of Lignin. In: Hofrichter M &Steinbüchel A, (ed), Lignin, Humic Substances and Coal, Vol 1, 2001, pp.89–116, Wiley-VHC, Weinheim.(45) Nimz, H. Beech lignin- Proposal of a constitutional scheme. Agnew. Chem.1974, 13(5), 313-321.149

5. Resultados y discusiónPublicación IV:Marques G., Gutiérrez A. and del Río J.C (2007) Chemical characterization oflignin and lipophilic fractions from leaf fibers of curaua (Ananas erectifolius).Journal of Agriculture and Food Chemistry, 55, 1327-1336.150

5. Resultados y discusiónChemical characterization of lignin and lipophilic fractions from leaf fibersof curaua (Ananas erectifolius)Gisela Marques, Ana Gutiérrez and José C. del RíoInstituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, P.O. Box 1052, 41080-Seville, SpainAbstractThe chemical composition of leaf fibers of curaua (Ananas erectifolius), anherbaceous plant native of Amazonia, was studied. Special attention was paid tothe content and composition of lignin and lipophilic compounds. The analysis oflignin in the curaua fibers was performed “in situ” by pyrolysis-gaschromatography/mass spectrometry (Py-GC/MS) and showed a lignincomposition with a p-hydroxyphenyl:guaiacyl:syringyl units (H:G:S) molarproportion 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 wasrevealed upon pyrolysis in the presence of tetramethylammonium hydroxide. Onthe other hand, the main lipophilic compounds, analysed by GC/MS, were seriesof long-chain n-fatty acids, n-fatty alcohols, - and -hydroxyacids,monoglycerides, sterols and waxes. Other compounds, such as -hydroxymonoesters and -hydroxy acylesters of glycerol were also found in this fiber inhigh amounts.Keywords: Curaua; Ananas erectifolius; lipids; lignin; pyrolysis; hydroxymonoesters; glyceryl esters; paper pulp.1. IntroductionAn alternative to wood raw materials for pulp and paper production indeveloping countries is the use of non-woody fibers from herbaceous fieldcrops. In developed countries non-woody fibers are mainly used for theproduction of specialty papers, i.e. tea bags, filter papers, bank notes, etc. Themain sources of non-woody raw materials are agricultural residues frommonocotyledons, including cereal straw and bagasse. Bamboo, reeds and someother grass plants such as flax, hemp, kenaf, jute, sisal or abaca are also grownor collected for the pulp industry but increased attention has been paid in recentyears to find new non-wood raw materials for pulp production.Curaua (Ananas erectifolius), an herbaceous plant native of the Amazonianregion and member of the bromeliad family, has been recognized since pre-Columbian days for its valuable fibers. In the last decade, it has gainedcommercial 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 is151

5. Resultados y discusiónbeing now investigated as an alternative lignocellulosic material for theproduction of chemical pulps.Studies on the chemical composition of curaua fibers are important toevaluate this fiber as a potential raw material for pulp and papermaking,however only limited studies have been performed so far on this interesting fiber[1-4]. In this work, we have performed a chemical characterization of curauafibers, paying especial attention to the content and composition of the lipophiliccompounds and the structural characterization of lignin, since these two organicfractions are of high importance during pulping and papermaking. It is knownthat the efficiency of pulping is directly proportional to the amount of syringyl(S) units in lignin [6, 7]. This is because the S-lignin is mainly linked by morelabile ether bonds, is relatively unbranched and has lower condensation degreethat G-lignin [8, 9]. Indeed, the S-lignin has higher reactivity in alkaline systemsthan G-lignin [10]. On the other hand, the lipophilic compounds present in rawmaterials cause significant environmental and technical problems in themanufacturing of paper pulp. During pulping, lipids are released from the fibersforming colloidal pitch, which can deposit in either pulp or machinery causingproduction troubles [11-13]. Moreover, such extractives might also contribute tothe toxicity of paper pulp effluents and products [14, 15].In the present study, the lignin in curaua fibers was characterized “in situ” byusing analytical pyrolysis coupled to gas chromatography/mass spectrometry(Py-GC/MS), which is a powerful analytical tool for the rapid analysis ofcomplex polymer mixtures, including lignocellulosic materials [16, 17].Pyrolysis in the presence of a methylating reagent, tetramethylammoniumhydroxide (TMAH), was used for the analysis of p-hydroxicinnamic acids (pcoumaricand ferulic acids). On the other hand, the lipid composition in curauafibers was analyzed by gas chromatography (GC) and gas chromatography/massspectrometry (GC/MS), using short- and medium-length high-temperaturecapillary columns, respectively [18], which enable the elution and analysis ofintact high molecular weight lipids such as waxes, sterol esters, andtriglycerides.2. Material and methods2.1. SamplesCuraua (Ananas erectifolius) fibers were supplied by CELESA pulp mill(Tortosa, Spain). The dried samples were milled using a knife mill (Janke andKunkel, Analysenmühle). For the isolation of lipids, the milled samples wereextracted with acetone in a Soxhlet apparatus for 8 h. The acetone extracts wereevaporated to dryness and weighted. Then, the extracts were resuspended inchloroform for chromatographic analysis of the lipophilic fraction. Tworeplicates were used for each sample, and all samples were subjected to GC andGC/MS analyses. For carbohydrate analysis and estimation of the Klason lignin152

5. Resultados y discusióncontent, the acetone extracted samples were subsequently extracted with hotwater (3 h at 100 ºC) to remove the water-soluble material. Holocellulose wasisolated from the pre-extracted fibers by delignification for 4 hours using theacid chlorite method (19). The -cellulose content was determined by removingthe hemicelluloses from the holocellulose by alkali extraction (19). Klasonlignin was estimated as the residue after sulfuric acid hydrolysis of the preextractedmaterial according to Tappi rule T222 om-88 [20]. The acid-solublelignin was determined, after filtering off the insoluble lignin, byspectrophotometric determination at 205 nm wavelength. Neutral sugars frompolysaccharide hydrolysis were analyzed as alditol acetates by GC according toTappi rule T249 om85 [20]. Ash content was estimated as the residue after 6 h at575 ºC. The general composition (as percent of whole fiber) was as follows:holocellulose, 92.5%; -cellulose, 66.4%; ash, 1.3%; acetone extractives, 5.3%;water-soluble extract, 5.1%; Klason lignin, 4.9%; acid-soluble lignin, 1.6%. Thecomposition of neutral monosaccharides (as percent of total neutralcarbohydrates) included arabinose, 2.7%; xylose, 8.0%; mannose, 3.5%;galactose, 0.2%; and glucose, 85.6%. No uronic acid determination wasperformed in this study. The composition of metals and other elements wasanalyzed by inductively coupled plasma spectrophotometry (ICP-OES) afteroxidation with concentrated HNO 3 under pressure in a microwave digestor, withthe 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.2.2. Solid Phase Extraction (SPE) fractionationFor a better characterization of the different homologous series, the lipid extractswere fractionated by a SPE procedure using aminopropyl-phase cartridges (500mg) from Waters Division of Millipore (Mildford, MA, USA), as alreadydescribed [18]. Briefly, the dried chloroform extracts were taken up in a minimalvolume (< 0.5 mL) of hexane:chloroform (4:1) and loaded into the cartridgecolumn previously conditioned with hexane (4 mL). The cartridge was loadedand eluted by gravity. The column was first eluted with 8 mL of hexane andsubsequently with 6 ml of hexane:chloroform (5:1), then with 10 mL ofchloroform and finally with 10 mL of diethyl ether:acetic acid (98:2). Eachisolated fraction was dried under nitrogen and analyzed by GC and GC/MS.2.3. GC and GC/MS analysesThe GC analyses of the extracts were performed in an Agilent 6890N NetworkGC system using a 5 m × 0.25 mm i.d., 0.1 m DB-5HT fused silica capillarycolumn from J&W Scientific (Folsom, CA, USA). The temperature programwas started at 100 ºC with a 1-min hold and then raised to a final temperature of350 ºC at 15 ºC/min, and held for 3 min. The injector and flame-ionizationdetector temperatures were set at 300 and 350 ºC, respectively. Helium was used153

5. Resultados y discusiónas the carrier gas at a rate of 5 mL/min, and the injection was performed insplitless mode. Peaks were quantified by area, and a mixture of standards(octadecane, palmitic acid, sitosterol and cholesteryl oleate) was used toelaborate calibration curves. The data from the two replicates were averaged. Inall cases the standard derivations from replicates were below 10% of the meanvalues.The GC/MS analyses were performed with a Varian model Star 3400 GCequipped with a model Saturn 2000 ion trap detector using a medium-length (12m) capillary column of the same characteristics described above. The oven washeated from 120 ºC (1 min) to 380 ºC at 10 ºC/min and held for 5 min. Thetransfer line was kept at 300 ºC. The injector was temperature programmed from120 ºC (0.1 min) to 380 ºC at a rate of 200 ºC/min and held until the end of theanalysis. Helium was used as the carrier gas at a rate of 2 mL/min. Methylationwith trimethylsilyldiazomethane and silylation withbis(trimethylsilyl)trifluoroacetamide (BSTFA) was used when required.Compounds were identified by comparing their mass spectra with mass spectrain Wiley and NIST libraries, by mass fragmentography, and when possible, bycomparison with authentic standards.2.4. Py-GC/MSThe pyrolysis of curaua fibers (approximately 100 g) was performed induplicate with a model 2020 micro-furnace pyrolyzer (Frontier LaboratoriesLtd., Yoriyama, Japan) directly connected to an Agilent 6890 GC/MS systemequipped with a 30 m × 0.25 mm i.d., 0.25 m HP 5MS fused silica capillarycolumn. The detector consisted of an Agilent 5973 mass selective detector (EI at70 eV). The pyrolysis was performed at 500 °C. The final temperature wasachieved at a rate of 20 °C/min. The GC/MS conditions were as follows: oventemperature was held at 50 °C for 1 min and then increased up to 100 °C at30 °C/min, from 100 to 300 °C at 10 °C/min and isothermal at 300 °C for10 min. The carrier gas used was helium with a controlled flow of 1 ml/min. Forthe pyrolysis in the presence of TMAH, approximately 100 g of sample wasmixed with 0.5 L of 25% TMAH. The pyrolysis was carried out as describedabove. The compounds were identified by comparing the mass spectra obtainedwith those of the Wiley and NIST computer libraries and that reported in theliterature [16, 17]. Relative peak molar areas were calculated for carbohydrateand lignin pyrolysis products. The summed molar areas of the relevant peakswere normalized to 100%, and the data for two repetitive pyrolysis experimentswere averaged. The relative standard deviation for the pyrolysis data was lessthan 5%.154

5. Resultados y discusión3. Results and discussionThe curaua fiber was characterized by a high holocellulose and -cellulosecontents (92.5 and 66.4, respectively), and a low lignin content (6.5% of thetotal fiber weight). This lignin content is similar to other non-wood fibers suchas flax or hemp and lower than other non-wood fibers such as kenaf or abaca[21-26]. The extractives content (5.3% of total fiber weight) is very high, andmuch higher than other nonwood fibers, which are usually less than 1% [21-26].However, most of the acetone extract corresponds to polar compounds, whileonly 1.3% corresponded to lipophilic compounds, which were estimated byredissolving the acetone extracts in chloroform. On the other hand, thehemicellulose fraction was mainly constituted by xylose. Finally, the ash content(1.3% of total fiber weight) was low in comparison to cereal straw [27], and thecomposition of the different metals revealed a predominance of Ca and K, and avery low content of other metals.3.1. Lignin compositionThe lignin composition of curaua fibers was analyzed “in situ” by Py-GC/MS.The Py-GC/MS chromatogram of curaua fibers is shown in Figure 1 and theidentities and relative molar abundances of the released compounds are listed inTable 1. The Py-GC/MS of curaua fibers released predominantly compoundsarising from carbohydrates, with only minor amounts of lignin-derived phenols.Carbohydrate pyrolysis products represented 88% on average and phenols fromlignin represented only 12% of the total identified compounds, which is inagreement with the low lignin content estimated as Klason lignin. Among thelignin derived compounds, the pyrogram of curaua fibers showed compoundsderived from p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) lignin units,with a slight predominance of the S units. The main lignin-derived compoundsidentified were 4-methylphenol (13), guaiacol (14), 4-vinylphenol (19), 4-vinylguaiacol (23), syringol (24), 4-ethylsyringol (33), 4-vinylsyringol (34) andtrans-4-propenylsyringol (40). The relative molar distribution of the differentlignin units (H:G:S) estimated by Py-GC/MS were 30:29:41 with a S/G molarratio of 1.4. The predominance of S-lignin observed in the curaua fiber isadvantageous for delignification during pulping because the S-lignin isrelatively unbranched and has lower condensation degree than H- and G-lignins.Moreover, S-lignin is more reactive in alkaline media [10].It must be noted that the relatively high abundances of 4-vinylphenol (19)observed in the pyrogram of curaua fibers could be due to the presence of p-coumaric acid, which upon pyrolysis will decarboxylate to produce 4-vinylphenol [28]. p-Hydroxycinnamic acids (p-coumaric and ferulic acids) occurwidely in the cell walls of herbaceous plants forming cross-linkages betweenlignin and polysaccharides [29-34]. The presence of p-hydroxycinnamic acidsconstitutes a complication for lignin analyses by analytical pyrolysis since they155

5. Resultados y discusiónyield pyrolysis products similar to those of corresponding lignin units. However,this problem can be solved by the use of pyrolysis in the presence of TMAH(Py/TMAH), which prevents decarboxylation and releases intact p-hydroxycinnamic acids (as their methyl derivatives), in addition to differentlignin degradation products [28, 35-38].Py/TMAH of curaua fibers released significant amounts of the methylderivative of p-coumaric acid (25% of the lignin and cinnamic acids releasedproducts) as well as minor amounts of the methyl derivative of ferulic acid (5%of the lignin and cinnamic acids released products). p-Hydroxycinnamic acidsare present in curaua fiber in relatively high amounts (cinnamic acids/ligninratio of 0.4, estimated after Py/TMAH) and agrees with the relatively highcontent of 4-vinylphenol released by Py-GC/MS. Studies on maize [39], wheat[40] and other grasses including bamboo [41] revealed that p-coumaric acid isesterified at the -position of lignin side-chains, and predominantly to S units[41, 42]. Therefore, probably the major part of the p-coumaric acid in curauafibers also attaches at the -position of the lignin side-chain by ester bonds. Therelatively high content of p-hydroxycinnamic acids in curaua fibers would alsobe advantageous for pulping since ester bonds are easily cleaved during cooking.491611,128192351018713141516172021222324252627 2928 30 3132,333435 363738403941 42 43442 4 6 8 10 12 14 16 18 20 22Retention time (minutes)Figure 1. Py-GC/MS chromatogram of curaua fibers. The identities and relative molarabundances of the compounds are listed in Table 1.156

5. Resultados y discusiónTable 1. Identification and Relative Molar Abundances (%) of the Compounds Released afterPy-GC/MS of Curaua Fibers.No Compound Mass Fragments MW Origin %1 acetic acid 45/60 60 C 35.82 2-hydroxypropanal 43/74 74 C 3.13 (3H)-furan-2-one 55/84 84 C 3.14 1,3-hydroxydihydro-2-(3H)-furanone 58/102 102 C 6.05 (2H)-furan-3-one 55/84 84 C 1.46 2-furaldehyde 67/95/96 96 C 6.47 cyclopent-1-ene-3,4-dione 54/68/96 96 C 0.98 (5H)-furan-2-one 55/84 84 C 4.29 2,3-dihydro-5-methylfuran-2-one 55/69/98 98 C 8.510 4-hydroxy-5,6-dihydro-(2H)-piran-2-one 58/85/114 114 C 2.111 3-hydroxy-2-methyl-2-cyclopenten-1-one 55/85/112 112 C 1.112 2-hydroxy-3-methyl-2-cyclopenten-1-one 55/85/112 112 C 4.813 4-methylphenol 77/107/108 108 LH 0.714 guaiacol 81/109/124 124 LG 0.515 2 furoic acid, methyl ester 67/95/126 126 C 1.316 4-methylguaiacol 95/123/138 138 LG 0.117 3,4-dihydroxybenzaldehyde 81/109/137/138 138 L 0.518 catechol 64/81/92/110 110 L/C 1.019 4-vinylphenol 65/91/120 120 LH/pCA 2.220 5-hydroxymethyl-2-furaldehyde 69/97/109/126 126 C 1.921 3-methoxycatechol 60/97/125/140 140 L 0.322 4-ethylguaiacol 122/137/152 152 LG 0.223 4-vinylguaiacol 107/135/150 150 LG 1.124 syringol 111/139/154 154 LS 0.725 eugenol 131/149/164 164 LG 0.226 4-propylguaiacol 122/136/166 166 LG

5. Resultados y discusión3.2. Lipid compositionThe total lipid extract of the curaua fibers accounted for 1.3% of the total fiberweight. The extracts were analyzed by GC and GC/MS according to the methoddeveloped by Gutiérrez et al. [18]. The chromatogram of the curaua extracts (asmethyl ester and TMSi ether derivatives) is shown in Figure 2 and the detailedlist with the identities and abundances of the main compounds present aresummarized in Table 2. The main compounds identified were series of n-fattyacids, n-fatty alcohols, - and -hydroxyacids, monoglycerides, sterols andwaxes. Other series of high molecular weight compounds such as -hydroxymonoesters and -hydroxy acylesters of glycerol, as well as sterol esters andsterol glycosides, were also present in important amounts. The structures of themain lipophilic compounds identified in the curaua extract are shown in Figure3. The -hydroxy fatty acids both in free or esterified form (forming esters withboth fatty alcohols and glycerol), was the main series of compounds present inthe extracts.OHM 243,4OH 20Al 22OHM 26Al 24 OH 24OH 24 M 24 OH OH 22 2212M 26FA 18:2FA 24 FA 16:1+FA 22 FA 18:1FAFA 18FA 20 16M 28W 38SGOHM 28W 40 CGW 42CE SE5 10 15 20 25Retention time (minutes)Figure 2. GC/MS chromatogram of the methyl ester and TMSi ether derivative of the lipidextract from curaua fibers. FA (n) , n-fatty acid series; Al (n) , alcohol series; W (n) , wax series;OH (n) and OH (n) , and -hydroxy fatty acids series; M (n) , monoglyceride series;OHM (n) , -hydroxy acylesters of glycerol series; SG, sitosteryl 3-D-glucopyranoside; CG,campesteryl 3-D-glucopyranoside;1, campesterol; 2, ergostanol; 3, sitosterol; 4,stigmastanol; CE, campesterol ester; SE, sitosterol ester; n denotes the total carbon atomnumber.158

5. Resultados y discusiónTable 2. Composition and Abundance (mg/Kg) of Lipophilic Compounds in Curaua Fiberscompound mass fragments (m/z) MW abundancefatty acids 813.2n-tetradecanoic acid 73/117/145/285* 285* 4.3n-pentadecanoic acid 73/117/145/299* 299* 3.39-hexadecenoic acid 55/69/236/254 254 7.6n-hexadecanoic acid 60/73/129/256 256 162.59,12-octadecadienoic acid 67/81/280 280 23.09-octadecenoic acid 55/69/264 282 91.6n-octadecanoic acid 60/73/129/284 284 262.0n-eicosanoic acid 60/73/129/312 312 24.2n-docosanoic acid 60/73/129/340 340 138.2n-tetracosanoic acid 60/73/129/368 368 96.5-hydroxy fatty acids 1423.316-hydroxyhexadecanoic acid 311/343/359 # 374 # 20.218-hydroxyoctadecanoic acid 339/371/387 # 402 # 28.420-hydroxyeicosanoic acid 367/399/415 # 430 # 30.822-hydroxydocosanoic acid 395/427/443 # 458 # 235.024-hydroxytetracosanoic acid 423/455/471 # 486 # 367.026-hydroxyhexacosanoic acid 451/483/499 # 514 # 532.528-hydroxyoctacosanoic acid 479/511/527 # 542 # 119.230-hydroxytriacontanoic acid 507/539/555 # 570 # 90.2-hydroxy fatty acids 226.52-hydroxyeicosanoic acid 73/117/355* 472* 62.72-hydroxydocosanoic acid 73/117/149/383* 500* 19.92-hydroxytetracosanoic acid 73/117/411* 528* 79.22-hydroxyhexacosanoic acid 73/117/439* 556* 64.7fatty alcohols 552.4n-eicosanol 75/103/355* 370* 8.9n-docosanol 75/103/383* 398* 247.2n-tetracosanol 75/103/411* 426* 242.2n-hexacosanol 75/103/439* 454* 44.3n-octacosanol 75/103/467* 482* 9.8sterols 618.7campesterol 55/145/213/382/400 400 56.9ergostanol 215/402 402 145.7sitosterol 145/213/396/414 414 226.4stigmastanol 215/416 416 189.7tocopherols 31.4-tocopherol 165/205/430 430 31.4steroid hydrocarbons 119.4ergostatriene 135/143/380 380 23.8159

5. Resultados y discusiónergostadiene 81/147/367/382 382 14.4stigmastadiene 81/147/381/396 396 71.8stigmasta-3,5,22-triene 135/143/394 394 2.6stigmasta-3,5-diene 81/147/381/396 396 6.8steroid ketones 57.9stigmasta-3,5-dien-7-one 174/269/410 410 7.7stigmast-4-en-3-one 124/229/412 412 24.4stigmastadienone isomer 57/136/174/269/410 410 16.4stigmastane-3,6-dione 245/287/428 428 9.4sterol esters 89.4campesterol ester 49.3sitosterol ester 40.1steryl glycosides 264.9campesteryl 3-Dglucopyranoside204/217/361/383* 850* 141.3sitosteryl 3-D-glucopyranoside 204/217/361/397* 864* 123.6waxes 173.2C 36 201/229/257/285/536 536 3.5C 37 243/257/550 550 3.3C 38 257/564 564 56.8C 39 243/257/271/285/299/578 578 5.9C 40 257/285/313/592 592 46.5C 40:1 264/283/590 590 2.9C 41 257/271/285/299/313/327/341/355/606 606 2.3C 42 257/285/313/341/620 620 24.9C 42:1 264/283/618 618 1.2C 43 257/271/285/299/313/327/355/369/634 634 1.5C 44 257/285/313/341/648 648 19.1C 46 257/285/313/341/369/397/676 676 5.3-hydroxy monoesters 369.5C 36 73/129/237/311/609* 624* 29.1C 37 73/129/237/311/623* 638* 3.0C 38 73/129/237/311/339/637* 652* 229.8C 40 73/129/237/311/339/367/395/665* 680* 95.6C 42 73/129/237/311/339/367/395/693* 708* 11.7C 44 73/129/367/395/721* 736* 0.3monoglycerides 714.61-monotetradecanoylglycerol 73/103/129/147/343/431* 446* 5.11-monohexadecanoylglycerol 73/103/129/147/371/459* 474* 5.31-monooctadecanoylglycerol 73/103/129/147/399/487* 502* 5.31-monoeicosanoylglycerol 73/103/129/147/427/515* 530* 31.61-monodocosanoylglycerol 73/103/129/147/455/543* 558* 288.21-monotetracosanoylglycerol 73/103/129/147/483/571* 586* 179.31-monohexacosanoylglycerol 73/103/129/147/511/599* 614* 168.2160

5. Resultados y discusión1-monooctacosanoylglycerol 73/103/129/147/539/627* 642* 23.41-monotriacontanoylglycerol 73/103/129/147/567/655* 670* 8.2-hydroxy acylesters of960.8glycerol1-mono(22-73/103/129/147/203/486/543/631* 646* 116.8hydroxydocosanoyl)glycerol1-mono(24-73/103/129/147/203/514/571/659* 674* 482.8hydroxytetracosanoyl)glycerol1-mono(26-73/103/129/147/203/542/599/687* 702* 301.7hydroxyhexacosanoyl)glycerol1-mono(28-73/103/129/147/203/570/627/715* 730* 59.5hydroxyoctacosanoyl)glyceroltr traces, * as TMSi ether derivates, # as methyl ester and TMSi ether derivates.Waxes (esters of fatty acids to fatty alcohols) were also important componentsof the curaua fiber extracts and were found in the range from C 36 to C 46 . Amongthe waxes, the GC/MS analysis revealed that each chromatographic peakconsisted of a complex mixture of different long-chain fatty acids esterified todifferent long-chain fatty alcohols. The identification and quantification of theindividual long-chain esters in each chromatographic peak was resolved basedon the mass spectra of the peaks. The mass spectra of long-chain esters arecharacterized by a base peak produced by a rearrangement process involving thetransfer of 2H atoms from the alcohol chain to the acid chain giving a protonatedacid ion [24, 43-45]. Therefore, the base peak gives the number of carbon atomsin the acid moiety and the molecular ion the total number of carbon atoms in theester. It is possible then to determine the individual contribution of the esters toevery chromatographic peak by mass spectrometric determination of themolecular ion and the base peak. Quantification of individual esters wasaccomplished by integrating areas in the chromatographic profiles of ionscharacteristic for the acidic moiety. The detailed structural composition andabundance of the high molecular weight waxes identified in the curaua fiber isshown in Table 3. The esterified fatty acids ranged from C 12 to C 25 and theesterified fatty alcohols ranged from C 16 to C 30 . Waxes with unsaturated fattyacids (C 40:1 and C 42:1 ) were also found in lower amounts, the unsaturated fattyacid being in all cases oleic acid.161

5. Resultados y discusiónTable 3. Composition of the Different Waxes (mg/kg) Identified in Curaua Fibers.Wax Fatty acid:fatty alcohol Abundancewax C 36 3.5C 12 :C 24 0.2C 14 :C 22 1.1C 16 :C 20 2.0C 18 :C 18 0.2wax C 37 3.3C 16 :C 21 0.9C 15 :C 22 2.4wax C 38 56.8C 16 :C 22 56.8wax C 39 5.9C 15 :C 24 0.9C 16 :C 23 2.6C 17 :C 22 2.2C 18 :C 21 0.2C 19 :C 20

C 18:1 :C 24 1.2wax C 43 1.5C 15 :C 28

5. Resultados y discusiónTable 4. Composition and abundance (mg/kg) of the different -hydroxy monoestersidentified in curaua fibers.-Hydroxy monoester -hydroxy fatty acid: fatty alcohol abundance-hydroxy monoester C 36 29.1-OHC 16 :C 20 29.1-hydroxy monoester C 37 3.0-OHC 16 :C 21 3.0-hydroxy monoester C 38 229.8-OHC 16 :C 22 223.9-OHC 18 :C 20 5.9-hydroxy monoester C 40 95.6-OHC 16 :C 24 59.5-OHC 18 :C 22 35.2-OHC 20 :C 20 0.9-hydroxy monoester C 42 11.7-OHC 16 :C 26 4.8-OHC 18 :C 24 0.7-OHC 20 :C 22 5.3-OHC 22 :C 20 0.9-hydroxy monoester C 44 0.3-OHC 20 :C 24 0.1-OHC 12 :C 22 0.2-Hydroxy fatty acids esterified to glycerol were also found in high amountsin the curaua fiber. The mass spectra of the TMSi derivatives of -hydroxyacylesters of glycerol are characterized by the presence of an abundant fragmentarising 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 103and [M-103] + . Other diagnostic ions are derived from the glyceryl moiety- i.e. atm/z 205 as a result of the cleavage between the C-2 and C-1 (the esterifiedcarbon), and at m/z 219 due to the loss of the acyloxy moiety. The same loss ofthe acyloxy group from M +· and M-15 + , but with the H rearrangement, gives riseto the ions at m/z 218 and 203, respectively. Other significant ions in the lowmassregion occur at m/z 73 (the TMSi group), m/z 129 (the glycerol carbonbackbone with a TMSi group [H 2 C=CHCH=O + Si(CH 3 ) 3 ]) and m/z 147(produced by the rearrangement of two TMSi groups) [51]. The -hydroxy fattyacids esterified to the glycerol ranges from C 22 to C 28 . The structure and massspectrum of the TMSi derivative of 1-mono-(22-hydroxydocosanoyl)glycerol isshown in Figure 5.164

5. Resultados y discusiónOAOHBOHHOCOOHODOHOHHOOOEOO-CH 2FHO-O-CHHO-O-CH 2HOOO-CH 2GHO-O-CHHO-O-CH 2HOHOHOHOH I J KCH 2 OHOCH 2 OHOHOOHOHOLHOOHOHOMFigure 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 3-D-glucopyranoside, M: sitosteryl 3-D-glucopyranoside.165

5. Resultados y discusión100%5573129[M-15] +[M-15-C 22 H 45 OH] +637311[C 15 H 31 COO-H 2 O] + [M-C 22 H 45 OH-H] +237327[M-TMSOH] + 343563100 200 300 400 500 600m/zFigure 4. Mass spectrum of trimethylsilylated hydroxy monoester C 38 .n-Fatty alcohols ranging from C 20 to C 28 were present in the curaua extractswith the presence of only the even carbon atom homologs, docosanol (C 22 ) andtetracosanol (C 24 ) being the most abundant. Monoglycerides, accounting for690.7 mg/Kg of the fibers, were present in important amounts, from C 14 to C 30 ,C 22 (1-monodocosanoylglycerol) being the most prominent. Di- andtriglycerides 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 ofminor amounts of stigmastanol, ergostanol and campesterol. Lower amounts ofsitosterol and campesterol could also be found in ester form. Sterol glycosides,such as sitosteryl and campesteryl 3-D-glucopyranosides were also identifiedin high amounts, the former being the most predominant. The identification ofsteryl glycosides was accomplished, after BSTFA derivatization of the lipidextract, by comparison with the mass spectra and relative retention times ofauthentic standards [52]. Finally, other compounds identified among the curauafiber extractives were -tocopherol, several steroid hydrocarbons and steroidketones, as reflected in Table 2.In conclusion, curaua fiber is characterized by a high content of holocelluloseand -cellulose and low lignin content which would make this fiber suitable forpapermaking. Moreover, the lignin composition indicates a slight predominanceof S-lignin units (S/G molar ratio of 1.4). On the other hand, the high extractivecontent can be considered as a detrimental aspect, however most of the acetoneextracts are due to polar compounds and only 1.3% corresponds to lipophiliccompounds. Indeed, most of the lipophilic compounds are easily saponifiable,and therefore can be hydrolyzed and dissolved during alkaline cooking.166

5. Resultados y discusión100%73OOOTMSOTMSTMSO129103147203265321[M-15] +381 411 [M-145] + [M-88] + 631486543100 200 300 400 500 600 m/zFigure 5. Mass spectrum and structure of the TMSi ether derivative of 1-mono(22-hydroxydocosanoyl)glycerol.AcknowledgementsThis study has been supported by the Spanish MEC (project AGL2005-01748).We thank CELESA (Tortosa, Spain) for providing the curaua fibers.Literature cited[1] Fujihashi, G. A.; Barbosa, W. L. R. Ananas erectifolius (curauá):padronização dos extractos, frações e do material vegetal. Revista Científica daUFPA. Vol 3, 2002 .[2] Silva, G. S.; Assis, M. B.; Barbosa, W. L. R. Investigação fitoquímica emicrobiologica da espécie Ananas erectifolius (curauá). Revista Virtual deIniciação Académica da UFPA. Vol 1, 2001.[3] Hoareau, W.; Trindade, W. G.; Siegmund B.; Castellan, A.; Frollini E. Sugarcane bagasse and curaua lignins oxidized by chlorine dioxide and reacted withfurfuryl alcohol: characterization and stability. Polym. Degrad. Stabil. 2004, 86,567-576.[4] Kelley, S. S.; Rowell, R. M.; Davis, M.; Jurich, Ch. K.; Ibach R. Rapidanalysis of the chemical composition of agricultural fibers using near infraredspectroscopy and pyrolysis molecular beam mass spectrometry. BiomassBioenergy. 2004, 27, 77-88.[5] Leao, A.L., Rowell, R., and Tavares, N., 1998. Applications of natural fibresin automotive industry in Brazil - Thermoforming process. In: (P.N. Prasad, J.E.167

5. Resultados y discusiónMark, S.H. Kandil, and Z.H. Kafafi, eds) Science and Technology of PolymersAdvanced Materials. Emerging technologies and business opportunities. Plenumpress, New York, pp 755-761.[6] González-Vila, F. J.; Almendros, G.; del Río, J. C; Martín, F.; Gutiérrez, A.;Romero, J. Ease of delignification assessment of wood from differentEucalyptus species by pyrolysis (TMAH)-GC/MS and CP/MAS 13 C NMRspectrometry. J. Anal. Appl. Pyrol. 1999, 49, 295-305.[7] del Río, J. C.; Gutiérrez, A.; Hernando, M.; Landín, P.; Romero, J.;Martínez, A. T. Determining the influence of eucalypt lignin composition inpaper 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.Int. Edit. 1974, 13, 313-321.[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 ofreactivity between syringyl lignin and guaiacyl lignin in alkaline systems.Holzforschung 1995, 49, 423-428.[11] Hillis, W. E.; Sumimoto, M. Effect of extractives on pulping. In NaturalProducts 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] Gutiérrez, A.; del Río, J. C.; Martínez, M. J.; Martínez, A. T. Thebiotechnological control of pitch in paper pulp manufacturing. TrendsBiotechnol. 2001, 19, 340-348.[14] Ali, M.; Sreekrishnan, T. R. Aquatic toxicity from pulp and paper milleffluents: A review. Adv. Environ. Res. 2001, 5, 175-196.[15] Rigol, A.; La Torre, A.; Lacorte, S.; Barceló, D. Bioluminiscenceinhibition assays for toxicity screening of wood extractives and biocides inpaper mill process waters. Environ. Toxicol. Chem. 2003, 23, 339-347.[16] Faix, O.; Meier, D.; Fortmann, I. Thermal degradation products of wood. Acollection of electron of electron-impact (EI) mass spectra of monomeric ligninderived products. Holz Roh- Werkst. 1990, 48, 351-354.[17] Ralph, J.; Hatfield, R. D. Pyrolysis-GC/MS characterization of foragematerials. J. Agric. Food Chem. 1991, 39, 1426-1437.168

5. Resultados y discusión[18] Gutiérrez, A.; del Río, J. C.; González-Vila, F. J.; Martín, F. Analysis oflipophilic extractives from wood and pitch deposits by solid-phase extractionand gas chromatography. J. Chromatogr. A. 1998, 823, 449-455.[19] Browning, B.L. Methods of Wood Chemistry, vol. II; Wiley-IntersciencePublishers, New York, USA, 1967.[20] Technical Association of the Pulp and Paper Industry. Test methods, 1992-1993. TAPPI, Atlanta, GA. 1993.[21] Gutiérrez, A.; Rodríguez, I. M.; del Río, J. C. Chemical characterization oflignin and lipid fractions in kenaf bast fibers used for manufacturing highqualitypapers. 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 fibersin textiles, pulp and paper production and composite materials. Directorate-General XII, Science, Research and Development, European Commision:Brussels, 1994.[23] del Río, J. C.; Gutiérrez, A. Chemical composition of abaca (Musa textilis)leaf fibers used for manufacturing of high quality paper pulps. J. Agric. FoodChem. 2006, 54, 4600-4610.[24] Gutiérrez. A.; del Río, J. C. Lipids from flax fibers and their fate in alkalinepulping. J. Agric. Food Chem. 2003, 51, 4965-4971.[25] Gutiérrez. A.; del Río, J. C. Lipids from flax fibers and their fate in alkalinepulping. J. Agric. Food Chem. 2003, 51, 6911-6914.[26] Gutiérrez, A.; Rodríguez, I. M.; del Río, J. C. Chemical characterization oflignin and lipid fractions in industrial hemp bast fibers used for manufacturinghigh-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 Río, J. C.; Martín, F.; González-Vila, F. J. Thermally assistedhydrolysis and alkylation as a novel pyrolytic approach for the structuralcharacterization of natural biopolymers and geomacromolecules. Trends Anal.Chem. 1996, 15, 70-79.[29] Lam, T. B. T.; Kadoya, K.; Liyama, K. Bonding of hydroxycinamic acidsto lignin: ferulic and p-coumaric acids are predominantly linked at the benzylposition of lignin, not the -position, in grass cell walls. Phytochemistry 2001,57, 987-992.169

5. Resultados y discusión[30] Sun, R. C.; Sun, X. F.; Zhang, S. H. Quantitative determination ofhydroxycinnamic acids in wheat, rice, rye, and barley straws, maize stems, oilpalm frond fiber, and fast-growing poplar wood. J. Agric. Food Chem. 2001, 49,5122-5129.[31] Grabber, J. H.; Ralph, J.; Hatfield, R. D. Cross-linking of maize walls byferulate dimerization and incorporation into lignin. J. Agric. Food Chem. 2000,48, 6106-6113.[32] Scalbert, A.; Monties, B.; Lallemand, J. Y.; Guittet, R.; Rolando, C. Etherlinkage between phenolic acids and lignin fractions from wheat straw.Phytochemistry 1985, 24, 1359-1362.[33] Sun, R. C.; Sun, X. F.; Wang, S. Q.; Zhu, W.; Wang, X. Y. Ester and etherlinkages between hydroxycinnamic acids and lignins from wheat, rice, rye, andbarley straws, maize stems, and fast-growing poplar wood. Ind. Crop Prod.2002, 15, 179-188.[34] Lam, T. B. T.; liyama, K.; Stone, B. A. Cinnamic acids bridges betweencell wall polymers in wheat and Phalaris internodes. Phytochemistry 1992, 31,1179-1183.[35] del Río, J. C.; McKinney, D. E.; Knicker, H.; Nanny, M. A.; Minard, R. D.Hatcher, P. G. Structural characterization of bio- and geo-macromolecules byoff-line thermochemolysis with tetramethylammonium hydroxide. J.Chromatogr. A 1998, 823, 433-448.[36] Martín, F.; del Río, J. C.; González-Vila, F. J.; Verdejo, T. Thermallyassisted hydrolysis and alkylation of lignins in the presence of tetraalkylammoniumhydroxides. J. Anal. Appl. Pyrol. 1995, 35, 1-13.[37] Challinor, J. M. Characterization of wood by pyrolysis derivatization-gaschromatography/ mass spectrometry. J. Anal. Appl. Pyrol. 1995, 35, 93-107.[38] Clifford, D. J.; Carson, D. M.; Mackinney. D. E.; Bortiatynski, J. M.;Hatcher, P. G. A new rapid technique for the characterization of lignin invascular plants: thermochemolysis with tetramethylammonium hydroxide(TMAH). Org. Geochem. 1995, 23, 169-175.[39] 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 byNMR. J. Chem. Soc. 1994, 34, 1-12.[40] Crestini, C.; Argyropoulos, D.S. Structural analysis of wheat straw ligninby quantitative 31 P and 2D NMR spectroscopy. The occurrence of ester bondsand -O-4 substructures. J. Agric. Food Chem. 1997, 45, 1212-1219.170

5. Resultados y discusión[41] Lu, F.C.; Ralph, J. Detection and determination of p-coumaroylated units inlignins. J. Agric. Food Chem. 1999, 47, 1988-1992.[42] Grabber, J.H.; Quideau, S.; Ralph, J. p-Coumaroylated syringyl units inmaize lignin: Implications for -ether cleavage by thioacidolysis.Phytochemistry 1996, 43, 1189-1194.[43] Sharkey, Jr. A. G.; Shultz, J. L.; Friedel, R. A. Mass spectra of esters.Formation of rearrangement ions. Anal. Chem. 1959, 31, 87-94.[44] Moldovan, Z.; Jover, E.; Bayona, J. M. Systematic characterization of longchainaliphatic esters of wool wax by gas chromatography-electron impactionisation mass spectrometry. J. Chromatogr. A 2002, 952, 193-204.[45] Reiter, B.; Lechner, M.; Lorbeer, E.; Aichholz, R. Isolation adcharacterization of wax esters in fennel and caraway seed oils by SPE-GC. J.High Res. Chromatogr. 1999, 22, 514-520.[46] Tulloch, A. P. The composition of beeswax and other waxes secreted byinsects. Lipids. 1969, 5, 247-258.[47] Tulloch, A. P. Beeswax: structure of the esters and their componenthydroxy acids and diols. Chem. Phys. Lipids. 1971, 6, 235-265.[48] Aichholz, R.; Lorbeer, E. Investigation of combwax of honeybees withhigh-temperature gas chromatography and high-temperature gaschromatography-chemical ionization mass spectrometry: I. High-temperaturegas chromatography. J. Chromatogr. A. 1999, 855, 601-615.[49] Aichholz, R.; Lorbeer, E. Investigation of combwax of honeybees withhigh-temperature gas chromatography and high-temperature gaschromatography-chemical ionization mass spectrometry: II. High-temperaturegas chromatography-chemical ionization mass spectrometry. J. Chromatogr. A.2000, 883, 75-88.[50] Kimpe, K.; Jacobs, P. A.; Waelkens, M. Mass spectrometric methods provethe use of beeswax and ruminant fat in late Roman cooking pots. J. Chromatogr.A. 2002, 968, 151-160.[51] Graça, J.; Schreiber, L.; Rodrigues, J.; Pereira, H. Glycerol and glycerylesters of -hydroxyacids in cutins. Phytochemistry. 2002, 61, 205-215.[52] Gutiérrez, A.; del Río, J. C. Gas chromatography/mass spectrometrydemonstration of steryl glycosides in eucalypt wood, kraft pulp and processliquids. Rapid Commun. Mass Spectrom. 2001, 15, 2515-2520.171

5. Resultados y discusiónPublicación V:Coelho D., Marques G., Gutiérrez A., Silvestre A.R.D. and del Río 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 discusiónChemical characterization of the lipophilic fraction of Giant reed (Arundodonax) fibres used for pulp and paper manufacturingDora Coelho 1,2 , Gisela Marques 1 , Ana Gutiérrez 1 , Armando J.D. Silvestre 2 , José C. del Río 11 Instituto de Recursos Naturales y Agrobiología, Consejo Superior de InvestigacionesCientíficas, P.O. Box 1052, 41080-Seville, Spain2 Departamento de Química, Universidade de Aveiro, 3810-193 Aveiro, PortugalAbstractThe 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, andthen fractionated by solid-phase extraction (SPE) on aminopropyl-phasecartridges into four different fractions of increasing polarity. The total lipidextract and the resulting fractions were analysed by gas chromatography and gaschromatography-mass spectrometry, using short- and medium-length hightemperaturecapillary columns, respectively. The main compounds identified inthe fibres included series of long-chain n-fatty acids, n-alkanes, n-aldehydes, n-alcohols, monoglycerides, free and esterified sterols and triterpenols, sterylglucosides, steroid hydrocarbons and steroid and triterpenoid ketones. Minoramounts of other compounds such as diglycerides, waxes and tocopherols werealso identified among the lipids of A. donax.Keywords: Arundo donax, lipophilic extractives, pitch, fatty acids, sterols, sterylglucosides, GC, GC/MS.1. IntroductionIn the last decades, fast growing plants have received particular attention asalternative sources of cellulose fibres (van Dam et al. 1994; Moore, 1996).These non-wood plants are the common fibre source for paper pulp productionin developing countries where wood fibres are not available. In the developedworld, although wood is still by far the main raw material for pulp and papermanufacture, a market exists for high-value-added papers from these fibres.Arundo donax L. (giant reed) is a widely distributed naturally growing perennialrhizomatous grass with a segmented tubular structure like bamboo (Seca et al.,2000), which has been considered as one of the promising non-wood plants forpulp and paper industry (Shatalov and Pereira, 2002). The easy adaptability todifferent ecological conditions, the annual harvesting period and the highbiomass productivity (32-37 t per year -1 ha -1 of dry biomass) reached by intensivecultivation (Vecchiet et al., 1996), combined with appropriate chemicalcomposition (Shatalov et al., 2001), make A. donax very attractive as analternative source of fibres (Shatalov and Pereira, 2005).173

5. Resultados y discusiónTo improve the utilisation of A. donax fibres, it is necessary to broaden theknowledge of structural features of its components. Previous chemical researchon A. donax includes chemical composition, general features of macromolecularcomponents (Pascoal Neto et al., 1997) and structures of isolated hemicelluloses(Driss et al., 1973, Joseleau and Barnoud, 1974, 1975, 1976). A few studies onthe lignin composition (Joseleau and Barnoud, 1976, Joseleau et al., 1976, Faixet al., 1989) showed that it is composed of guaiacyl- and syringyl-propane unitswith minor amounts of p-hydroxyphenylpropane units (Faix et al., 1989) andassociated with phenolic acids (Tai et al., 1987). However, until now no studiesabout the composition of A. donax lipophilic fraction have been performed.The amount and composition of lipophilic extractives is an importantparameter in wood processing for pulp and paper production and it is dependenton factors such as the plant species, age, and growth location. The different lipidclasses have different chemical behaviour during pulping and bleaching(Gutiérrez and del Río, 2003; Freire et al., 2005). The lipophilic extractives arealso responsible for the formation of sticky deposits on the machinery, givingrise to dark spots in bleached pulp and paper, the so-called pitch, both withnegative economic impact on pulp and paper industry (del Río et al., 1998,2000; Gutiérrez et al., 2004; Gutiérrez and del Río, 2005; Silvestre et al., 1999).The accumulation of lipophilic compounds leads also to higher chemicalconsumption during pulping and bleaching and therefore increasing productioncosts. On the other hand, extractives or their derivatives, might contribute to thetoxicity of paper pulp effluents and products (McCubbin and Folke 1995; Rigolet al., 2003). The detailed identification of such lipophilic components istherefore an important step in the study of the behaviour and fate of extractivesduring pulp and paper production and consequently in the search for newsolutions to control pitch deposition as well as to decrease effluent toxicity.In the present paper, the chemical composition of the lipophilic extractivesfrom A. donax fibres was studied. Gas chromatography (GC) and GC-massspectrometry (GC/MS) using, respectively, short- and medium-length hightemperaturecapillary columns with thin films, that enable elution and separationof high-molecular-mass lipids such as waxes, steryl esters and triglycerides, areemployed. For a more detailed characterization of the different homologousseries and other minor compounds, the extract was fractionated by a simplesolid-phase extraction (SPE) method using aminopropyl phase cartridges, asdescribed previously (Gutiérrez et al., 1998, 2004).2. Experimental2.1. SamplesSamples of A. donax L. reed stems (including nodes and internodes) weresupplied by University of Huelva, Spain. The samples were air-dried and milledusing a knife mill (Janke and Kunkel, Analysenmühle). For the isolation of174

5. Resultados y discusiónlipids, the milled samples were Soxhlet extracted with acetone for 8h. Thelipophilic extractives were obtained by redissolving the dried acetone extract inchloroform and evaporated to dryness under nitrogen.2.2. Solid Phase Extraction (SPE) fractionationThe chloroform extracts (5-20 mg) were fractionated by a SPE procedure inaminopropyl phase cartridges (500 mg) from Waters (Dvision of Millipore,Milford, MA, USA), as already described (Gutiérrez et al., 1998, 2004). Briefly,the dried extract was taken up in a minimal volume (

5. Resultados y discusiónmass spectra in Wiley and NIST libraries, by mass fragmentography, and, whenpossible, by comparison with authentic standards.3. Results and discussionThe total acetone extract from A. donax fibres accounted for 1.56% of total fibreweight. The lipophilic – chloroform soluble – compounds represented 0.62%,while the remaining 0.94% corresponded to polar compounds non-soluble inchloroform. The lipid extracts were analyzed by GC and GC/MS according tothe method previously described (Gutiérrez et al. 1998, 2004). The GC/MSchromatogram of the A. donax fibres extract, as trimethylsilyl (TMS)derivatives, is shown in Figure 1. For a better characterization of thecompounds present in the lipid extracts, these were subsequently fractionated bySPE in aminopropyl-phase cartridges into four major fractions of increasingpolarity. The chromatograms of the different SPE fractions are shown in Figure2. The first fraction (A), eluted with hexane, was enriched in steryl esters, waxesand hydrocarbons. The second fraction (B), eluted with hexane:chloroform(5:1), contained steroid ketones. The third fraction (C), eluted with chloroform,contained sterols, fatty alcohols and mono- and diglycerides. A final fraction (D)SitosterolFA28FA16Steryl glycosidesSGFA18:2FA18:1CampesterolStigmasterolFA30FA17FA18MG16FA20FA24MG18FA26MG26StGCGSteryl/triterpenyl esters5 10 15 2030Retention time (min)Figure 1. GC/MS chromatogram of the derivatized (TMS) chloroform extract of Arundodonax fibres. FA: fatty acids; MG: monoglycerides; CG: campesteryl 3-D-glucopyranoside;StG: stigmasteryl 3-D-glucopyranoside; SG: sitosteryl 3-D-glucopyranoside.176

5. Resultados y discusiónAk 27Ak 29ASteryl estersAk 25Ak 315 10 15 20 25CycloartenoneB-amyrenone-amyrenone5 10 15 20 25-Sitosterol + Stigmastanol + -amyrinCCampesterolStigmasterol-amyrin7-oxositosterol5 10 15 20 25FA28DFA16FA18:1+FA18:2FA18FA24FA265 10 15 20 25Figure 2. GC/MS chromatograms of the different SPE fractions isolated from the A. donaxfibres extracts. Fraction A, eluted with 8 mL of hexane; fraction B, eluted with 6mL ofhexane:chloroform (5:1); fraction C, eluted with 10 mL of chloroform; and fraction D, elutedwith 10 mL diethyl ether:acetic acid (98:2). FA: fatty acids; AK: n-alkanes.177

5. Resultados y discusiónTable 1. Chemical composition of lipophilic extractives in Arundo donax reed (mg/Kg offibre). Each value is the average of two extractions with variation coefficients within 0.1-4.5% .Compound Mass Fragments MW Amountn-Alkanes 77.9n-docosane 57/71/85/310 310 0.5n-tricosane 57/71/85/324 324 0.2n-tetracosane 57/71/85/338 338 0.6n-pentacosane 57/71/85/352 352 6.3n-hexacosane 57/71/85/366 366 3.9n-heptacosane 57/71/85/380 380 15.8n-octacosane 57/71/85/394 394 6.7n-nonacosane 57/71/85/408 408 37.0n-triacontane 57/71/85/422 422 0.8n-hentriacontane 57/71/85/436 436 5.4n-dotriacontane 57/71/85/450 450 0.3n-tritriacontane 57/71/85/464 464 0.4Steroid hydrocarbons 127.4ergostatriene 135/143/380 380 14.5ergostadiene 81/147/367/382 382 9.3estigmastadiene 81/147/381/396 396 8.4estigmasta-3,5,22-triene 135/143/394 394 49.2estigmasta-3,5-diene 81/147/381/396 396 46.0Fatty acids 1137.7n-tetradecanoic acid 73/117/132/145/285/300 * 300* 3.5n-pentadecanoic acid 73/117/132/145/299/314 * 314* 1.8n-hexadecanoic acid 60/73/129/256 256 276.3n-heptadecanoic acid 73/117/132/145/327/342 * 342 * 10.09,12-octadecadienoic acid 67/81/280 280 30.09-octadecanoic acid 55/69/264 282 55.7n-octadecanoic acid 60/73/129/284 284 73.6n-nonadecanoic acid 73/117/132/145/355/370 370* 3.1n-eicosanoic acid 60/73/129/312 312 50.0n-heneicosanoic acid 55/69/129/326 326 3.3n-docosanoic acid 60/73/129/340 340 35.7n-tricosanoic acid 60/73/129/354 354 25.3n-tetracosanoic acid 60/73/129/368 368 55.7n-pentacosanoic acid 60/73/129/382 382 33.5n-hexacosanoic acid 73/117/132/145/453/468 * 468 * 144.1n-heptacosanoic acid 73/117/132/145/467/482 482* 14.3n-octacosanoic acid 73/117/132/145/482/497 * 497 * 134.9n-nonacosanoic acid 73/117/132/145/495/510 * 510 * 53.9n-triacontanoic acid 73/117/132/145/509/525 * 525 * 109.9n-hentriacontanoic acid 73/117/132/145/523/538 538* 6.2n-dotriacontanoic acid 73/132/145/117/537/552 * 552 * 16.9Fatty alcohols 194.3n-hexacosanol 75/103/439* 454* 33.4178

5. Resultados y discusiónn-octacosanol 75/103/467* 482* 54.9n-triacontanol 75/103/495* 510* 57.7n-dotriacontanol 75/103/523* 538* 48.3Aldehydes 81.6n-hexacosanal 82/96/362 380 10.4n-octacosanal 82/96/390 408 22.9n-triacontanal 82/96/418 436 48.3Sterols/Triterpenols 528.1campesterol 55/145/213/382/400 400 90.6stigmasterol 55/81/255/394/412 412 46.4sitosterol 145/213/396/414 414 281.0stigmastanol 215/416 416 71.97-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 23.5Tocopherol 17.7-tocopherol 151/416 416 6.8-tocopherol 165/430 430 10.9Triterpenoid and steroid ketones 43.9-amyrenone 189/203/218/409/424 424 10.2-amyrenone 189/203/218/409/424 424 5.9cycloartenone 189/205/313/409/424 424 14.2stigmasta-3,5-dien-7-one 174/269/410 410 3.2stigmast-4-en-3-one 124/229/412 412 4.6stigmast-4-en-3,6-dione 137/398/408/411/426 426 3.6stigmastane-3,6-dione 245/287/428 428 2.5Steryl /triterpenyl esters 68.1sitosteryl ester 147/381/397 - 16.1-amyrinyl ester 189/203/218 - 14.0-amyrinyl ester 189/203/218 - 38.0Steryl glucosides 151.6campesteryl 3--D-glucopyranoside 204/217/361/383 * 850 * 30.6stigmasteryl 3--D-glucopyranoside 204/217/361/395 * 864 * 8.0sitosteryl 3--D-glucopyranoside 204/217/361/397 * 862 * 113.0Monoglyceride 367.52,3-dihydroxypropyl tetradecanoate 73/103/129/147/343/431 * 446 * 5.52,3-dihydroxypropyl hexadecanoate 73/103/129/147/371/459 * 474* 94.22,3-dihydroxypropyl octadecanoate 73/103/129/147/399/487 * 502 * 86.62,3-dihydroxypropyl eicosanoate 73/103/129/147/427/515 * 530 * 35.12,3-dihydroxypropyl docosanoate 73/103/129/147/455/543 * 558 * 43.02,3-dihydroxypropyl tetracosanoate 73/103/129/147/483/571 * 586 * 46.92,3-dihydroxypropyl hexacosanoate 73/103/129/147/511/599 * 614 * 56.2179

5. Resultados y discusiónDiglycerides 47.6dipalmitin, 1,2- (P2) 57/129/313/386/625 * 640 * 7.8dipalmitin, 1,3- (P2) 57/129/314/371/385/625 * 640 * 12.1palmitoylstearin (PS) 57/129/314/372/399/579 * 668 * 16.8distearin, 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). Theidentities and abundances of the main compounds identified are listed in Table1. The most predominant lipid classes identified among the A. donax lipidextracts 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, steroidand triterpenoid ketones and steryl/triterpenyl esters, were also present in thesefibres. The structures of main and representative compounds are shown inFigure 3.The series of free fatty acids were identified in A. donax fibres ranging fromtetradecanoic (C14) to dotriacontanoic (C32) acids, with strong even-over-oddcarbon atom predominance. Hexadecanoic acid (palmitic acid, I) was the mostabundant fatty acid, however a bimodal distribution, with a second maximumfor octacosanoic acid (C28) was observed. The unsaturated 9-octadecenoic(oleic acid, II) and 9,12-octadecadienoic (linoleic acid, III) acids were alsopresent in important amounts. The series of n-alkanes was also identified in theA. donax fibre ranging from docosane (C22) to tritriacontane (C33), with astrong odd-over-even carbon atom number predominance, and nonacosane (IV)being the most predominant homolog. n-Fatty alcohols ranging fromhexacosanol (C26) to dotriacontanol (C32) were present in the A. donax extractswith 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 identifiedin the A. donax fibres with triacontanal (VI) predominating. Monoglycerideswere also present in high amounts in A. donax fibres. The series ofmonoglycerides was identified in the range from C14 to C26, with maximum formonopalmitin, C16, (VII).Steroids and triterpenoids, including free sterols, steryl esters, steryl glucosides,steroid ketones and hydrocarbons are among the most predominant compoundsin the lipophilic extract of A. donax fibre. Free sterols were the major compoundclass among steroids and triterpenoids, sitosterol (VIII) being the main sterolpresent. Other sterols, such as campesterol (IX), stigmasterol (X), stigmastanol(XI) and the oxidized 7-oxositosterol, were also present. Steryl esters were alsopresent in A. donax extract, although in low amounts. The completeidentification of the individual steryl esters by GC-MS was not possible sincethey only show fragments arising from the sterol moiety by electro-impact MS180

5. Resultados y discusiónFigure 3. Structures of the main lipophilic compounds present in A. donax fibres. (I) palmiticacid, (II) oleic acid, (III) linoleic acid, (IV) nonacosane, (V) triacontanol, (VI) triacontanal,(VII) monopalmitin, (VIII) sitosterol, (IX) campesterol, (X) stigmasterol, (XI) stigmastanol,(XII) sitosteryl 3-D-glucopyranoside, (XIII) -amyrin, (XIV) -amyrin, (XV) stigmasta-3,5-diene, (XVI) stigmasta-3,5,7-triene, (XVII) -amyrenone, (XVIII) -amyrenone, (XIX)cycloartenone, (XX) stigmasta-3,5-dien-7-one, (XXI) stigmast-4-en-3-one, (XXII) stigmasta-3,6-dione.181

5. Resultados y discusiónand rarely give detectable molecular ions (Lusby et al. 1984, Evershed et al.1989). By monitoring the ions corresponding to the different sterol moieties inthe SPE fraction enriched in steryl esters, it was possible to identify series ofsitosterol as well as - and -amyrin esters. Steryl glucosides, such ascampesteryl, stigmasteryl and sitosteryl -D-glucopyranosides (XII), wereidentified in significant amounts, the latter being the most predominant.The identification of steryl glucosides was accomplished (after BSTFAderivatization of the lipid extract) by comparison with the mass spectra andrelative retention times of authentic standards (Gutiérrez and del Río, 2001).Among triterpenols, -amyrin (XIII) and -amyrin (XIV) occurred in free andesterified form, with the latest being detected in low amounts. Finally, severalsteroid hydrocarbons, such as stigmasta-3,5-diene (XV) and stigmasta-3,5,7-triene (XVI) and triterpenoid and steroid ketones, such as -amyrenone (XVII),-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 differentbehavior during pulping and bleaching and therefore the problematic of pitchwill be different depending the type of pulping (i.e. mechanical, chemical) andbleaching (ECF, TCF) processes. The knowledge of the chemical compositionof the lipophilic components of A. donax fibres shown here will assist to predictpitch problems during pulp and papermaking of this fibre and to establishappropriate methods for their control.AcknowledgementsThis study has been funded by the Spanish project AGL2005-01748. GM thanksthe Spanish Ministry of Education and Science for a FPI fellowship. We thankM.J. Diaz (University of Huelva) for the Arundo donax fibres.Referencesdel Río, J.C., Gutiérrez A., Gonz.lez-Vila F.C., Martín F. and Romero J., 1998.Characterization of organic deposits produced in kraft pulping of Eucalyptusglobulus wood. J. Chromatogr. A. 823, 457-465.del Río, J.C.; Romero, J.; Gutiérrez, A., 2000. Analysis of pitch depositsproduced 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 propertiesof two xylans of reed (Phragmites communis and Arundo donax) in solution.Cell. Chem. Technol. 7, 703-713.182

5. Resultados y discusiónEvershed, R.P., M.C. Prescott, N. Spooner and L.J. Goad. 1989. Negative ionammonia chemical ionization and electron impact ionization massspectrometric analysis of steryl fatty acyl esters. Steroids 53, 285–309.Faix, O., Meier, D., Beinhoff, O., 1989. Analysis of lignocelluloses and ligninsfrom Arundo donax and Miscanthus sinensis Anderss and hydroliquefactionof Miscanthus. Biomass 18, 109.Freire, C.S.R., Silvestre, A.J.D. and Pascoal Neto, C., 2005. Lipophilicextractives in Eucalyptus globulus Kraft pulps. Behaviour during ECFbleaching. J. Wood Chem. Technol. 25, 67-80.Gutiérrez, A., del Río, J.C., González-Vila F.J. and Martín F., 1998. Analysis oflipophilic extractives from wood and pitch deposits by solid-phase extractionand gas chromatography. J. Chromatogr. A. 823, 449-455.Gutiérrez, A.; del Río, J.C., 2001. Gas chromatography/mass spectrometrydemonstration of steryl glycosides in eucalypt wood, Kraft pulp and processliquids. Rapid Commun. Mass Spectrom., 15, 2515-2520.Gutiérrez, A.; del Río, J. C., 2003. Lipids from flax fibers and their fate inalkaline pulping. J. Agric. Food Chem., 51, 4965-4971.Gutiérrez, A., del Río, J.C. and Martínez, A.T., 2004. Chemical Analysis andBiological Removal of Wood Lipids forming Pitch deposits in paper pulpmanufacturing. 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.Gutiérrez, A. and del Río, J.C., 2005. Chemical characterization of pitchdeposits produced in the manufacturing of high-quality paper pulps fromhemp fibers. Biores. Technol. 96, 1445-1450.Joseleau, J.P. and Barnoud, F., 1974. Hemicelluloses of young internodes ofArundo donax. Phytochemistry 13, 1155-1158.Joseleau, J.P. and Barnoud, F., 1975. Hemicelluloses of Arundo donax atdifferent stages of maturity. Phytochemistry 14, 71-75.Joseleau, J.P. and Barnoud, F., 1976. Cell wall carbohydrates and structuralstudies 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 ofArundo donax lignin in relation to growth. Holzforschung 31, 19-20.183

5. Resultados y discusiónLusby, W. R., M. J. Thompson and J. Kochansky. 1984. Analysis of sterol estersby capillary gas chromatography electron impact and chemical ionizationmassspectrometry. Lipids 19(11), 888–901.McCubbin, N. and J. Folke. 1995. Significance of AOX vs. unchlorinatedorganics. Pulp Paper Can. 96, 43-48.Moore, G., 1996. Nonwood Fibre Applications in Papermaking; PiraInternational, Leatherhead, Surrey, UK.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 chemicalcomposition and structure of macromolecular components in differentmorphological regions and maturity stages of Arundo donax. Ind. Crops Prod.6, 51-58.Rigol, A.; La Torre, A.; Lacorte, S.; Barceló, D., 2003. Bioluminiscenceinhibition assays for toxicity screening of wood extractives and biocides inpaper mill process waters. Environ. Toxicol. Chem., 23, 339-347.Seca, A.M., Cavaleiro, J.A.S., Domingues, F.M.J., Silvestre, A.J.D, Evtuguin,D., Neto, C.P., 2000. Structural characterization of the lignin from the nodesand internodes of Arundo donax Reed. J.Agric.Food Chem. 48, 817-824.Shatalov, A.A., Quilhó, T., Pereira, H., 2001. Arundo donax L. reed – newperspectives 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 andpaper properties of Arundo donax L. reed. Ind. Crops Prod. 15, 77-83.Shatalov, A.A., Pereira, H., 2005. Kinetics of organosolv delignification of fibrecrop Arundo donax L. Ind. Crops Prod. 21, 203-210.Silvestre, A. J.D., C. L.C. Pereira, C. Pascoal Neto, A.C. Duarte, J. A. S.Cavaleiro and F. P. Furtado. 1999. Chemical composition of pitch depositsfrom an ECF Eucalyptus globulus bleached kraft pulp mill: Its relationshipwith wood extractives and additives in process streams. Appita J. 52(5), 375-382.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 applicationof domestically produced plant fibres in textiles, pulp and paper production184

5. Resultados y discusiónand composite materials. Directorate-General XII, Science, Research andDevelopment, European Commission.Vecchiet, M., Jodice, R., Schenone, G., 1996. Agronomic research on giant reed(Arundo donax L.). Management system and cultivation of two differentprovenances. 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. 644-648.185

5. Resultados y discusiónPublicación VI:Marques G., Gutiérrez A. and del Río J.C. (2008) Chemical composition oflignin and lipids from tagasaste (Chamaecytisus proliferus spp. palmensis).Industrial Crops and Products, 28, 29-36.186

5. Resultados y discusiónChemical composition of lignin and lipids from tagasaste (Chamaecytisusproliferus spp. palmensis)Gisela Marques, Ana Gutiérrez and José C. del RíoInstituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, P.O. Box 1052, 41080-Seville, SpainAbstractThe chemical characterization of trimming residues of tagasaste (Chamaecytisusproliferus spp. palmensis), a hardy leguminous shrub that has been recentlyexplored for pulp and paper production, was performed with especial emphasisin the composition of lignin and lipophilic extractives. Tagasaste wascharacterized by a high content of holocellulose (81%) and -cellulose (41%),while having a relatively low lignin content of 18.9%. The analysis of lignin wasperformed “in situ” by pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) and showed a composition with a guaiacyl:syringyl (G:S) molarproportion 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 makestagasaste an adequate raw material for pulping. On the other hand, the relativelyhigh acetone extractive content (1.4%) was mostly due to polar compounds andonly 0.2% corresponded to lipophilic compounds. The lipophilic compounds,analyzed by GC and GC/MS, were mainly composed of fatty acids, including -hydroxyfatty acids, and steroid compounds, such as free and conjugated (estersand glycosides) sterols.Keywords: Tagasaste; lipids; lignin; pyrolysis; paper pulp.1. IntroductionTagasaste (Chamaecytisus proliferus spp. palmensis), also known as “treelucerne”, is a hardy leguminous and fast-growing shrub of the Fabaceae(Genisteae) family. It is indigenous of the Canary Islands (Spain) but is nowbeing cultivated in Australia, New Zealand and other countries (Francisco-Ortega et al., 1991). The shrub is being mainly exploited for high-protein fodderto maintain livestock (Borens and Poppi, 1990; Ventura et al., 2002) and also asN-fixing crops to improve soil fertility (Kindu et al., 2006). In order toencourage the formation of bushes with multiple stems the shrub must be grazedwith regularity, which leads to a high accumulation of trimming residues. Theseresidues are nowadays considered as agricultural waste since they cannot beconverted to valuable products.As attempts to reduce the adverse environmental impact and to use thisrenewable biomass, they have been recently explored as an alternative rawmaterial for pulp production (Díaz et al. 2004; López et al., 2004; Jiménez et al.,187

5. Resultados y discusión2006; Jiménez et al., 2007; García et al., 2007). Tagasaste has been found to bean excellent raw material for paper pulp production, similar to eucalypt wood,with high hollocellulose and -cellulose contents, and low lignin and extractivescontent, giving high yields (Díaz et al. 2004; López et al., 2004; Jiménez et al.,2006). However, despite all this previous work, a detailed chemical compositionof tagasaste trimming residues has not been addressed so far, which is of highimportance for optimizing the use of this raw material for paper pulp production.The content and chemical structure of woody components, in particular thelignin content and its composition in terms of its p-hydroxyphenyl (H), guaiacyl(G) and syringyl (S) moieties are important parameters in pulp production inview of delignification rates, chemical consumption and pulp yields. The higherreactivity of the S lignin with respect to the G lignin in alkaline systems isknown (Chang and Sarkanen 1973; Tsutsumi et al. 1995) and therefore, thelignin S/G ratio in hardwoods affects the pulping efficiency. It has already beenshown for eucalypt woods that higher S/G ratios imply higher delignificationrates, less alkali consumption and therefore higher pulp yield (González-Vila etal. 1999; del Río et al. 2005).On the other hand, the composition of extractives, especially the lipophiliccompounds, is also important for pulp and paper production. The differentclasses of lipids have different behavior during cooking and bleaching. Thelipids can be classified into two principal groups, namely fatty acids and neutralcomponents, the latter including waxes, long chain n-fatty alcohols, alkanes andsteroids and triterpenoids. And the behavior of the fatty acids in an aqueousenvironment is quite different from that of the neutrals. In alkaline pulping, theacids dissociate and can dissolve in water to quite a high extent, forming fattyacid soaps. The neutrals, however, have a very low solubility in water andsurvive the cooking process and remain in the pulp being at the origin of the socalledpitch deposits, which are responsible of reduced product quality andhigher operating costs due to production stops for cleaning the equipment (Hillisand Sumimoto 1989; Back and Allen 2000). The increasing trend inrecirculating water in pulp mills to accomplish environmental demands isaggravating these problems.Therefore, the main objective of this work is to perform a thorough chemicalcharacterization of tagasaste trimming residues, with especial emphasis in thechemical composition of lignin and lipophilic extractives. In this work, thelignin composition of tagasaste was characterized “in situ” using analyticalpyrolysis coupled to gas chromatography/mass spectrometry (Py-GC/MS), apowerful analytical tool for the rapid analysis of complex polymer mixturesincluding lignocellulosic materials (Ralph and Hatfield 1991; Faix et al. 1990;del Río et al. 2001; del Río et al. 2005) that can give information on the lignincomposition in terms of the H, G and S moieties. On the other hand, thechemical characterization of the lipophilic extractives was performed by GC andGC/MS by using high-temperature short- and medium-length capillary columns,188

5. Resultados y discusiónrespectively. This method enables the elution and analysis of intact highmolecular weight lipids (Gutiérrez et al. 1998). The knowledge of the chemicalcomposition of the main components of tagasaste trimming residues will beuseful for a better utilization of this agricultural waste.2. Experimental2.1. SamplesTagasaste trimming residues were supplied by University of Huelva, Spain. Thedried samples were milled using a knife mill. For the isolation of lipids, themilled samples were extracted with acetone in a Soxhlet apparatus for 8 h. Theacetone extracts were evaporated to dryness and resuspended in chloroform forchromatographic analysis of the lipophilic fraction. Two replicates were used foreach sample, and all samples were subjected to GC and GC/MS analyses. ForKlason lignin content estimation, the samples extracted with acetone weresubsequently extracted with hot water (3 h at 100 ºC) to remove the watersolublematerial. Holocellulose was isolated from the pre-extracted fibers bydelignification for 4 hours using the acid chlorite method (Browning, 1967). The-cellulose content was determined by removing the hemicelluloses from theholocellulose by alkali extraction (Browning, 1967). Klason lignin wasestimated as the residue after sulfuric acid hydrolysis of the pre-extractedmaterial according to Tappi rule T222 om-88 (Tappi, 1993). The acid-solublelignin was determined, after filtering off the insoluble lignin, byspectrophotometric determination at 205 nm wavelength. Ash content wasestimated as the residue after 6 h at 575 ºC.2.2. Solid Phase Extraction (SPE) fractionationFor a better characterization of the different homologous series, the lipid extractswere fractionated by a SPE procedure using aminopropyl-phase cartridges (500mg) from Waters (Division of Millipore), as already described (Gutiérrez et al.1998). Briefly, the dried chloroform extracts were taken up in a minimal volume(< 0.5 ml) of hexane:chloroform (4:1) and loaded into the cartridge columnpreviously conditioned with hexane (4 ml). The cartridge was loaded and elutedby gravity. The column was first eluted with 8 ml of hexane and subsequentlywith 6 ml of hexane:chloroform (5:1), then with 10 ml of chloroform and finallywith 10 ml of diethyl ether:acetic acid (98:2). Each isolated fraction was driedunder nitrogen and analyzed by GC and GC/MS.2.3. GC and GC/MS AnalysesThe GC analyses of the extracts were performed in an Agilent 6890N GCsystem using a short-fused silica capillary column (DB-5HT, 5 m × 0.25 mmI.D., 0.1 m film thickness). The temperature program was started at 100 ºC189

5. Resultados y discusiónwith a 1-min hold and then raised to a final temperature of 350 ºC at 15 ºC/min,and held for 3 min. The injector and flame-ionization detector temperatures wereset at 300 and 350 ºC, respectively. Helium was used as the carrier gas at a rateof 5 mL/min, and the injection was performed in splitless mode. Peaks werequantified 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 GCequipped with an ion trap detector (Varian Saturn 2000) using a medium-length(12 m) capillary column of the same characteristics described above. The ovenwas heated from 120 (1 min) to 380 ºC at 10 ºC/min and held for 5 min. Thetransfer line was kept at 300 ºC. The injector was temperature programmed from120 (0.1 min) to 380 ºC at a rate of 200 ºC/min and held until the end of theanalysis. 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 producethe appropriate derivatives, when required. Compounds were identified bycomparing their mass spectra with mass spectra in Wiley and NIST libraries, bymass fragmentography, and when possible, by comparison with authenticstandards.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 systemAgilent 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 5973mass selective detector. The pyrolysis was performed at 500 °C. The finaltemperature was achieved at a rate of 20 °C/min. The GC/MS conditions were asfollows: oven temperature was held at 50 °C for 1 min and then increased up to100 °C at 30 °C/min, from 100 to 300 °C at 10 °C/min and isothermal at 300 °Cfor 10 min using a heating rate of 20 °C/min in the scan modus. The carrier gasused was helium with a controlled flow of 1 ml/min. The compounds wereidentified by comparing the mass spectra obtained with those of the Wiley andNIST computer libraries and that reported in the literature (Faix et al. 1990;Ralph and Hatfield 1991). Relative peak molar areas were calculated forcarbohydrate and lignin pyrolysis products. The summed molar areas of therelevant peaks were normalized to 100%, and the data for two repetitivepyrolysis experiments were averaged.3. Results and DiscussionTagasaste was characterized by a high content of holocellulose (81%) and -cellulose (41%), and a lignin content of 18.9% (Klason lignin, 16.6%; acidsolublelignin: 2.3%). Similar values have been reported by other authors (Díaz190

5. Resultados y discusiónet 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, andtogether with the high holocellulose and -cellulose contents makes tagasaste aninteresting raw material for pulp and paper production, as already advanced byother authors (Díaz et al. 2004; López et al., 2004; Jiménez et al., 2006; Jiménezet al. 2007; García et al., 2007). On the other hand, the acetone extractivesaccounted 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 (Backand Allen, 2000). However, while the content of the main organic fractions is animportant parameter in wood processing for pulp and papermaking, the chemicalcomposition of these organic fractions, in particular the lignin composition interms of the relative proportions of the S- and G-units, and the lipidcomposition, in terms of the presence of saponifiable or unsaponifiablecomponents, strongly influences the pulping and bleaching behavior of a rawmaterial.3.1. Lignin compositionThe lignin composition of tagasaste was characterized in situ by Py-GC/MS.The pyrogram of tagasate sample is shown in Figure 1 and the identities andrelative molar abundances of the released compounds are listed in Table 1. Py-GC/MS of tagasaste wood trimming residues released compounds arising fromcarbohydrates (67%) and lignin-derived phenols (34%). The lignin-derivedphenols arise from guaiacyl (G) and syringyl (S) lignin units whereas no tracesof p-hydroxyphenyl (H) units were found in tagasaste. The main lignin-derivedcompounds identified were 4-vinylsyringol (38), syringol (24), 4-methylsyringol(29), 4-ethylsyringol (35) and 4-vinylguaiacol (23). Syringaldehyde (41),syringylacetone (47) and trans-sinapaldehyde (49) were also identified. Relativepeak molar areas were calculated for carbohydrate, and lignin G- and S-typedegradation products. The Py-GC/MS data showed a lignin composition intagasaste with a predominance of the S units (S/G molar ratio of 1.6), typical ofhardwoods. The predominance of S-lignin units in tagasaste makes this materialadvantageous for delignification due to the higher reactivity of the S-lignin inalkaline systems (Chang and Sarkanen, 1973; Tsutsumi et al. 1995; González-Vila et al., 1999) and its lower condensation degree. The G units have a free C-5position available for carbon-carbon inter-unit bonds, which make them fairlyresistant to lignin depolymerization in pulping, while the S lignin is relativelyunbranched and has a lower condensation degree and therefore is easier todelignify. The high S/G ratio, together with its low lignin content, explains theexcellent performances of tagasaste during alkaline pulping.191

5. Resultados y discusiónTable 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 39.42 (3H) furan-3-one C 55/84 84 0.73 1,3-hydroxydihydro-2-(3H)-furanone C 58/102 102 3.14 (3H) furan-3-one C 55/84 84 1.05 2-furaldehyde C 67/95/96 96 3.86 2-(hydroxymethyl)-furan C 43/70/81/98 98 0.57 cyclopent-1-ene-3,4-dione C 54/68/96 96 0.68 (5H) furan-2-one C 55/84 84 2.99 2,3-dihydro-5-methylfuran-2-one C 55/69/98 98 3.110 5-methyl-2-furfuraldehyde C 53/109/110 110 0.411 4-hydroxy-5.6-dihydro-(2H)-pyran-2-one C 58/85/114 114 2.612 3-hydroxy-2-methyl-2-cyclopenten-1-one C 55/85/112 112 0.613 2-hydroxy-3-methyl-2-cyclopenten-1-one C 55/85/112 112 0.314 dihydroxypyran-1-one C 56/84/114 114 0.815 guaiacol LG 81/109/124 124 1.916 dimethyldihydropyranone C 55/70/83/111/126 126 0.317 4-methylguaiacol LG 95/123/138 138 1.218 catechol C 64/81/92/110 110 1.119 5-hydroxymethyl-2-tetrahydrofuraldehyde-3-one C 57/69/85/98/99 138 0.420 5-hydroxymethyl-2-furaldehyde C 69/97/109/126 126 0.321 methoxycatechol L 60/97/125/140 140 0.922 4-ethylguaiacol LG 122/137/152 152 0.423 4-vinylguaiacol LG 107/135/150 150 2.124 syringol LS 111/139/154 154 3.625 eugenol LG 131/149/164 164 0.426 4-propylguaiacol LG 122/136/166 166 0.127 vanillin LG 109/151/152 152 0.728 cis-isoeugenol LG 131/149/164 164 0.429 4-methylsyringol LS 125/153/168 168 1.830 trans-isoeugenol LG 131/149/164 164 1.231 homovanillin LG 122/137/166 166 0.232 4-propinylguaiacol LG 77/91/119/147/162 162 1.033 4-propinylguaiacol isomer LG 77/91/119/147/162 162 1.334 acetovanillone LG 123/151/166 166 0.235 4-ethylsyringol LS 167/182 182 1.536 guaiacylacetone LG 122/137/180 180 0.737 levoglucosane C 60/98 162 4.838 4-vinylsyringol LS 137/165/180 180 4.239 4-allylsyringol LS 167/179/194 194 0.740 cis-4propenylsyringol LS 167/179/194 194 0.541 syringaldehyde LS 167/181/182 182 1.2192

5. Resultados y discusión42 4-propynylsyringol LS 106/131/177/192 192 1.043 4-propynylsyringol isomer LS 106/131/177/192 192 0.844 trans-4-propenylsyringol LS 167/179/194 194 2.545 acetosyringone LS 153/181/196 196 0.646 trans-coniferaldehyde LG 107/135/147/178 178 0.847 syringylacetone LS 123/167/210 210 0.548 propiosyringone LS 151/181/210 210 0.349 trans-sinapaldehyde LS 137/165/180/208 208 1.3C: cellulose; LG: lignin guaiacyl; LS: lignin syringyl%G 38%S 62S/G 1.63.2. Lipid compositionThe lipid composition in tagasaste was analyzed by gas chromatography (GC)and gas chromatography/mass spectrometry (GC/MS), using short- andmedium-length high-temperature capillary columns, respectively (Gutiérrez etal., 1998), which enable the elution and analysis of intact high molecular weightlipids such as waxes, sterol esters, and triglycerides. The GC/MS chromatogramof the lipids from tagasaste is shown in Figure 2. The identities and abundances138245442398111513610 1416171821 222325 27 2829303233 35 363941424045 4647 48492 4 6 8 10 12 14 16 18 20 22Retention time (min)Figure 1. Py-GC/MS of tagasaste wood. The identities of the peaks are shown in Table 2.193

6 7 8 sterol esters5. Resultados y discusiónof the main lipid classes identified are summarized in Table 2. The structures ofthe main lipophilic compounds identified in the tagasaste extracts are shown inFigure 3. The main lipids identified in tagasaste were series of fatty acids,including -hydroxy acids, and steroid compounds, including steroidhydrocarbons, steroid ketones, sterols, sterol esters and sterol glycosides. Othercompounds, such as series of alkanes and monoglycerides were also found inminor amounts.The series of free fatty acids (141.4 mg/kg) were present in the range from n-tetradecanoic (C 14 ) to n-hexacosanoic (C 26 ) acids, with a strong even-over-oddcarbon atom predominance. Hexadecanoic (palmitic) acid (I, C 16:0 ) andoctadecanoic (stearic) acid (C 18:0 ) were the most abundant fatty acids followedby n-tetracosanoic (C 24 ) acid. Unsaturated fatty acids were also present, 9-octadecenoic (oleic) acid (II, C 18:1 ) being especially abundant. Fatty acids alsoincluded a series of -hydroxyfatty acids (51.8 mg/kg) that was present in therange from 2-hydroxyoctadecanoic acid (C 18 ) to 2-hydroxyhexacosanoic acid(C 26 ) with maximum at C 24 (III), and the presence of exclusively the evencarbon atom number homologs.34+5steroid 2hydrocarbonssteroidketonesFA 16FA 18:1FA 18FA 20FA 22 FA 2415 10 15 20Retention time (min)Figure 2. GC/MS of the lipophilic extracts from tagasaste wood. Key labels are: F n : fattyacids; 1: -tocopherol; 2: campesterol; 3: stigmasterol; 4: sitosterol; 5: stigmastanol; 6: 7-oxocampesterol; 7: 7-oxostigmasterol; 8: 7-oxositosterol.194

5. Resultados y discusiónTable 2.- Composition and abundance (mg/Kg) of lipophilic extractives in tagasaste trimmingresidues.compound mass fragments MW abundancen-alkanes 8.8n-eicosane 57/71/85/282 282 0.1n-heneicosane 57/71/85/296 296 0.2n-docosane 57/71/85/310 310 0.1n-tricosane 57/71/85/324 324 0.3n-tetracosane 57/71/85/338 338 0.2n-pentacosane 57/71/85/352 352 0.5n-hexacosane 57/71/85/366 366 0.3n-heptacosane 57/71/85/380 380 0.5n-octacosane 57/71/85/394 394 0.3n-nonacosane 57/71/85/408 408 1.2n-triacontane 57/71/85/422 422 0.2n-hentriacontane 57/71/85/436 436 4.8n-tritriacontane 57/71/85/464 464 0.1fatty acids 141.4n-tetradecanoic acid 60/73/228 228 1.1n-pentadecanoic acid 60/73/242 242 1.09-hexadecenoic acid 55/69/236/254 254 2.2n-hexadecanoic acid 60/73/129/256 256 55.4n-heptadecanoic acid 60/73/129/270 270 2.99-octadecenoic acid 55/69/264 282 29.4n-octadecanoic acid 60/73/129/284 284 10.7n-nonadecanoic acid 60/73/129/298 298 1.4n-eicosanoic acid 60/73/129/312 312 7.4n-heneicosanoic acid 60/73/129/327 327 1.1n-docosanoic acid 60/73/129/340 340 5.4n-tricosanoic acid 60/73/129/354 354 6.0n-tetracosanoic acid 60/73/129/368 368 10.3n-pentacosanoic acid 60/73/129/382 382 2.6n-hexacosanoic acid 60/73/129/396 396 4.5-hydroxy fatty acids # 51.82-hydroxyoctadecanoic acid 73/327/371 386 0.62-hydroxyeicosanoic acid 73/355/399 414 0.92-hydroxydocosanoic acid 73/383/427 442 22.12-hydroxytetracosanoic acid 73/411/455 470 26.32-hydroxyhexacosanoic acid 73/439/483 498 1.9steroid hydrocarbons 30.1ergostatriene 135/143/380 380 5.6ergostadiene 81/147/367/382 382 3.4stigmasta-3,5,22-triene 135/143/394 394 11.7195

5. Resultados y discusiónstigmasta-3,5-diene 81/147/381/396 396 9.4sterols 113.1campesterol 55/145/213/382/400 400 14.0stigmasterol 55/83/255/394/412 412 35.7sitosterol 145/213/396/414 414 30.7stigmastanol 215/416 416 2.97-oxocampesterol 382/397/414 414 10.17-oxostigmasterol 286/426 426 9.07-oxositosterol 396/413/428 428 10.7-tocopherol 165/205/430 430 2.1steroid ketones 30.5stigmasta-7,22-dien-3-one 55/269/298/367/410 410 7.1stigmasta-3,5-dien-7-one 174/269/410 410 14.1stigmast-4-en-3-one 124/229/412 412 4.5stigmastadienone isomer 57/136/174/269/410 410 2.0stigmastane-3,6-dione 245/287/428 428 2.8sterol glycosides* 13.2campesterol 3-D-glucopyranoside 204/217/361/383 850 1.9stigmasterol 3-D-glucopyranoside 204/217/361/413 880 4.3sitosterol 3-D-glucopyranoside 204/217/361/397 864 7.0sterol esters 28.3campesterol esters n.d 2.3stigmasterol esters n.d 9.2sitosterol esters n.d 16.8monoglycerides* 8.11-monopalmitin 73/103/129/147/371/459 474 4.21-monostearin 73/103/129/147/399/487 502 3.9* as TMS ethers; # as methyl esters and TMS ethersSteroid compounds were the second most important lipid class found amongthe tagasaste extractives and included steroid hydrocarbons, steroid ketones andsterols (in free and conjugated form). Sterols, in free and conjugated (esters andglycosides) form were the most important steroids identified, free sterols beingpresent in important amounts (113.1 mg/kg). Stigmasterol (IV) was the mostabundant among the free sterols with the presence of important amounts ofcampesterol (V) and sitosterol (VI) and minor amounts of stigmastanol (VII) aswell as the presence of the oxidized 7-ketocampesterol, 7-ketostigmasterol and7-ketostigmastanol. Lower amounts of sterols could also be found in ester form(28.3 mg/kg), with a predominance of sitosterol esters. Sterol glycosides, suchas campesteryl, stigmasteryl and sitosteryl 3-D-glucopyranosides (VIII) were196

5. Resultados y discusiónFigure 3. Structures of the main lipophilic compounds present in tagasaste. I: hexadecanoicacid; II: 9-hexadecenoic acid; III: 2-hydroxyhexacosanoic acid; IV: stigmasterol; V:campesterol; VI: sitosterol; VII: stigmastanol; VIII: sitosteryl 3-D-glucopyranoside; IX:stigmasta-3,5-dien-7-one; X: stigmasta-3,5,22-triene; XI: n-hentriacontane; XII: 1-monopalmitin.also identified in lower amounts (13.2 mg/kg), the latter being the mostpredominant. The identification of steryl glycosides was accomplished, afterBSTFA derivatization of the lipid extract, by comparison with the mass spectraand relative retention times of authentic standards (Gutiérez and del Río, 2001).It is important to point out the low content of free and conjugated (esters andglycosides) sterols, in comparison to that found in eucalypt wood (Rencoret etal., 2007), since these are the main compounds responsible for pitch depositionduring kraft cooking of hardwoods, such as eucalypt wood (del Río et al. 1998,2000; Silvestre et al. 1999; Gutiérrez and del Río 2001). On the other hand,197

5. Resultados y discusiónsteroid ketones were also found in important amounts (30.5 mg/kg), beingmainly 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 (30.1mg/kg), stigmasta-3,5,22-triene (X) being the most predominant.Finally, a series of n-alkanes ranging from n-eicosane (C 20 ) to n-tritriacontane(C 33 ) with n-hentriacontane (X, C 31 ) being the most prominent, was found in lowamounts (8.8 mg/kg). And minor amounts of monoglycerides (8.1 mg/kg), suchas 1-monopalmitin (XI) and 1-monostearin, were also present among tagasasteextractives.4. ConclusionsTagasaste is characterized by a high content of holocellulose and -celluloseand a relatively low lignin content. Moreover, the chemical composition oftagasaste lignin indicates a predominance of S-lignin units over the G-ligninones (S/G molar ratio of 1.6) and the absence of H-lignin units. The totalacetone extractives accounted for 1.4% but the content on lipophilic compoundsis very low (only 0.2%). The main lipids identified in tagasaste were series offatty acids, including -hydroxyfatty acids, and steroid compounds, includingsteroid hydrocarbons, steroid ketones, sterols, sterol esters and sterol glycosides.AcknowledgementsThis study has been supported by the Spanish project AGL2005-01748. G.Marques acknowledges a FPI Fellowship from the Spanish Ministry ofEducation and Science. We thank Manuel J. Díaz (University of Huelva, Spain)for providing the tagasaste samples.ReferencesBack, E.L., Allen, L.H., 2000. Pitch Control, Wood Resin and Deresination.Tappi Press, Atlanta.Borens, F., Poppi, D.P., 1990. The nutritive and feeding value for ruminants oftagasaste (Chamaecytisus palmensis), a leguminous tree. Anim. Feed Sci.Technol. 28, 275–292.Browning, B.L., 1967. Methods of Wood Chemistry, vol. II; Wiley-IntersciencePublishers, New York.Chang, H-M., Sarkanen, K.V., 1973. Species variation in lignin. Effect ofspecies on the rate of kraft delignification. Tappi 56, 132-134.198

5. Resultados y discusióndel Río, J.C., Gutiérrez, A., González-Vila, F.J., Martín, F., Romero, J., 1998.Characterization of organic deposits produced in the Kraft pulping ofEucalyptus globulus wood. J. Chromatogr. A 823, 457-465.del Río, J.C., Romero, J., Gutiérrez, A., 2000. Analysis of pitch depositsproduced in Kraft pulp mills using a totally chlorine free bleaching sequence.J. Chromatogr. A 874, 235-245.del Río J.C., Gutiérrez A., Romero J., Martínez M.J. and Martínez A.T., 2001.Identification of residual lignin markers in eucalypt kraft pulp by Py-GC-MS.J. Anal. Appl. Pyrolysis 58/59, 425-439.del Río, J.C., Gutiérrez, A., Hernando, M., Landín, P., Romero, J., Martínez,A.T., 2005. Determining the influence of eucalypt lignin composition in paperpulp yield using Py-GC/MS. J. Anal. Appl. Pyrolysis 74, 110-115.Díaz, M.J., Alfaro, A., García, M.M., Eugenio, F.M.E., Ariza, J., López, 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 monomericlignin derived products. Holz Roh- Werkst. 48, 351-354.Francisco-Ortega, J., Jackson, M.T., Santos-Guerra, A., Fernández-Galván, M.,1991. Historical aspects of the origin and distribution of tagasaste(Chamaecytisus proliferus (L. fil.) Link ssp. palmensis (Christ) Kunkel), afodder tree from the Canary Islands. J. Adelaide Botanical Garden 14, 67–76.García, M.M., López, F., Alfaro, A., Ariza, J., Tapias, R., 2007. The use oftagasaste (Chamaecytisus proliferus) from different origins for biomass andpaper production. Biores. Technol. (in press).González-Vila, F.J., Almendros, G., del Río, J.C., Martín, F., Gutiérrez, A.,Romero, J. 1999. Ease of delignification assessment of wood from differentEucalyptus species by pyrolysis (TMAH)-GC/MS and CP/MAS 13 C NMRspectrometry. J. Anal. Appl. Pyrol. 49, 295-305.Gutiérrez, A., del Río, J.C., González-Vila, F.J., Martín, F., 1998. Analysis oflipophilic extractives from Wood and pitch deposits by solid-phase extractionand gas chromatography. J. Chromatogr. A 823, 449-455.199

5. Resultados y discusiónGutiérrez, A., del Río, J.C., 2001. Gas chromatography/mass spectrometrydemonstration of steryl glycosides in eucalypt wood, kraft pulp and processliquids. Rapid Commun. Mass Spectrom. 15, 2515-2520.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.Jiménez, L., Pérez, A., Rodríguez, A., de La Torre, M.J., 2006. New rawmaterials and pulping processes for production of pulp and paper. Afinidad 63(525), 362-369.Jiménez, L., Pérez, A., de la Torre, M.J., Moral A., Serrano, L., 2007.Characterization of vine shoots, cotton stalks, Leucaena leucocephala andChamaecytisus proliferus, and of their ethyleneglycol pulps. Biores. Technol.98, 3487-3490.Kindu, M., Glatzel, G., Tadesse, Y., Yosef, A., 2006. Tree species screened onnitosols of central Ethiopia: Biomass production, nutrient contents and effecton soil nitrogen. J. Trop. Forest Sci. 18, 173-180.López, F., Alfaro, A., García, 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.Ralph, J., Hatfield, R.D., 1991. Pyrolysis-GC/MS characterization of foragematerials. J. Agric. Food Chem. 39, 1426-1437.Rencoret, J., Gutiérrez, A., del Río, J.C., 2007. Lipid and lignin composition ofwoods 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 pitchdeposits from an ECF Eucalyptus globulus bleached kraft pulp mill: itsrelationship 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 ofreactivity between syringyl lignin and guaiacyl lignin in alkaline systems.Holzforschung 49, 423-428.200

5. Resultados y discusiónVentura, M.R., Castanon, J.I.R., Rey, L., Flores, M.P., 2002. Chemicalcomposition and digestibility of tagasaste (Chamaecytisus proliferus)subspecies for goats. Small Ruminant Res. 46, 207-210.201

5. Resultados y discusiónPublicación VII:Marques G., del Río J.C. and Gutiérrez A. (2010) Lipophilic extractives fromseveral nonwoody lignocellulosic crops (flax, hemp, sisal, abaca) and their fateduring alkaline pulping and TCF/ECF bleaching. Bioresource Technology, 101,260-267.202

5. Resultados y discusiónLipophilic extractives from several nonwoody lignocellulosic crops (flax,hemp, sisal, abaca) and their fate during alkaline pulping and TCF/ECFbleachingGisela Marques, José C. del Río and Ana GutiérrezInstituto de Recursos Naturales y Agrobiología, CSIC, PO Box 1052, E-41080, Seville, SpainAbstractThe fate of lipophilic extractives from several nonwoody species (flax, hemp,sisal and abaca) used for the manufacturing of cellulose pulps, was studiedduring soda/anthraquinone (AQ) pulping and totally chorine free (TCF) andelemental chlorine free (ECF) bleaching. With this purpose, the lipophilicextracts from the raw materials and their unbleached and bleached industrialpulps, were analyzed by gas chromatography-mass spectrometry. Aldehydes,hydroxyfatty acids and esterified compounds such as ester waxes, sterol estersand alkylferulates strongly decreased after soda/AQ pulping while alkanes,alcohols, free sterols and sterol glycosides survived the cooking process. Amongthe lipophilic extractives that remained in the unbleached pulps, some amountsof free sterols were still present in the TCF pulps whereas they were practicallyabsent in the ECF pulps. Sterol glycosides were also removed after both TCFand ECF bleaching. By contrast, saturated fatty acids, fatty alcohols and alkaneswere still present in both bleached pulps.Keywords: pitch; lipophilic extractives; paper pulp; nonwoody fibers1. IntroductionLipophilic extractives, i.e. the non-polar extractable fraction from wood andother lignocellulosic crops, include different classes of compounds, such asalkanes, fatty alcohols, fatty acids, free and conjugated sterols, terpenoids,triglycerides and waxes. These lipophilic compounds, even when present in lowamounts in the raw material, may play an important role in industrial woodprocessing for bleached pulp and paper production since they are at the origin ofthe 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 isresponsible for reduced production levels, higher equipment maintenance costs,higher operating costs, and an increased incidence of defects in the finishedproducts, which reduces quality and benefits (Back and Allen, 2000).The nature and severity of pitch deposition depend not only on the rawmaterials used (and hence on the nature of the lipophilic compounds), but alsoon the industrial processes of pulping and bleaching applied at the mill. In themanufacture of alkaline pulps, a large part of the lipids originally present in the203

5. Resultados y discusiónraw 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; Gutiérrez et al.,2001a), suspended in process waters (Gutiérrez et al., 2001b) or forming the socalledpitch deposits in circuits, equipments and final product (Bergelin et al.,2005; del Río et al., 1998; 2000; Gutiérrez and del Río, 2005; Silvestre et al.,1999). Growing pressure for closing water loops in pulp and paper mills leads toincreasing build up of lipophilic compounds in the processes and therefore, to anincreasing number of severe and costly pitch related problems.The different classes of lipids have different behavior during pulping andbleaching. Several studies have provided information on the behavior of woodlipids, such as fatty and resin acids, triglycerides and sterols and triterpenolsduring pulping and bleaching (Back and Allen, 2000; Bergelin and Holmbom,2003; 2008; Freire et al., 2005; 2006; Gutiérrez et al., 2001a; Shin and Kim,2006). However, the chemistry of lipids from nonwoody pulps in pulping andbleaching has not been examined much so far (Gutiérrez et al., 2004; Gutiérrezand del Río, 2003a; 2003b). Likewise, to the best of our knowledge, there isvery little information available dealing with pitch problems on nonwoody pulps(Gutiérrez and del Río, 2005). In this context, the aim of this work was toidentify the specific lipophilic constituents of different nonwoody fibers, whichare used for the manufacturing of cellulose pulps for specialty papers, and tostudy their behavior during soda/anthraquinone (AQ) pulping and totally chorinefree (TCF) and elemental chlorine free (ECF) bleaching. For this, a series ofpulps (crude pulps taken after soda/AQ pulping and bleached pulps taken afterTCF and ECF bleaching) from different nonwoody raw materials (flax, hemp,sisal, abaca) were selected for this study. The composition of the lipophiliccompounds in the fibers and their respective pulps was analyzed by gaschromatography (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 intacthigh molecular weight lipids such as waxes or sterol glycosides (Gutiérrez et al.,1998; Gutiérrez and del Río, 2001). The knowledge of the behavior of lipophilicextractives during pulping and bleaching is an important step towardsunderstanding and predicting the pitch problems and designing effectivesolutions for its control.2. Materials and methods2.1. SamplesTwo bast fibers, flax (Linum usitatissimum) and hemp (Cannabis sativa), andtwo leaf fibers, sisal (Agave sisalana) and abaca (Musa textilis), were selectedfor this study. The raw materials and their respective crude (unbleached) pulps(pulps taken after soda/AQ pulping), as well as pulp samples after TCF and ECF204

5. Resultados y discusiónbleaching, were supplied by CELESA pulp mill (Tortosa, Spain). Generalconditions of soda/AQ pulping included the use of sodium hydroxide andanthraquinone (up to 0.05%) as cooking chemicals, and 2-3 h of cooking time ata temperature of about 165ºC. These general conditions can be slightly modifieddepending on the raw material used. The TCF bleaching sequence used (QPoP)included a quelating stage (Q) and two hydrogen peroxide stages (P), the firstone under pressurized oxygen (Po). The ECF bleaching sequence used (DPo)included a chlorine dioxide stage (D) followed by a hydrogen peroxide stageunder pressurized oxygen (Po).2.2. Lipid extractionRaw materials and pulps were air-dried until constant weight and the sampleswere Soxhlet-extracted with acetone for 8 h. All the extracts were evaporated todryness and redissolved in chloroform for chromatographic analysis of thelipophilic fraction as described below.2.3. GC and GC-MS analysesThe GC analyses of the lipids from the raw materials and pulps were performedin an Agilent 6890N Network GC system using a short fused-silica DB-5HTcapillary column (5 m x 0.25 mm internal diameter, 0.1 μm film thickness) fromJ&W Scientific, enabling simultaneous elution of the different lipid classes(Gutiérrez et al., 1998). The temperature program was started at 100°C with 1min hold, and then raised to 350°C at 15°C/min, and held for 3 min. The injectorand flame-ionization detector (FID) temperatures were set at 300°C and 350°C,respectively. Helium (5 ml/min) was used as carrier gas, and the injection wasperformed in splitless mode.The GC-MS analyses were performed with a Varian 3800 chromatographcoupled to an ion-trap detector (Varian 4000) using a medium-length (12 m)capillary column of the same characteristics described above for GC/FID. Theoven 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 programmed from 120°C(0.1 min) to 380°C at 200°C/min and held until the end of the analysis, andhelium was used as carrier gas at a rate of 2 ml/min. The compounds wereidentified by mass fragmentography, and by comparing their mass spectra withthose of the Wiley and NIST libraries and standards.Both underivatized and derivatized samples were analyzed by GC and GC-MS. Two derivatized samples, silylated samples and methylated and silylatedsamples, were analyzed. The silylation was peformed usingbis(trimethylsilyl)trifluoroacetamide (BSTFA) in the presence of pyridine at80ºC for 90 min (Gutiérrez and del Río, 2001). Trimethylsilyl-diazomethanemethylation, in the presence of methanol (at room temperature for 10 min)followed by BSTFA silylation, in the presence of pyridine, were also performed.205

5. Resultados y discusiónFatty alcohols, fatty acids and sterol glycosides were determined as silylatedderivatives in the derivatized samples. Hydroxyfatty acids were determined inthe samples after methylation followed by silylation. The rest of compoundswere determined in the underivatized samples. Peaks were quantified by areaand a mixture of standards (octadecane, palmitic acid, sitosterol, cholesteryloleate, and sitosteryl 3-D-glucopyranoside) with a concentration range between0.1 and 1mg/ml was used to elaborate calibration curves. The data from the tworeplicates were averaged.3. Results and discussionThe lipid content of the nonwoody fibers used in this study and theircorresponding crude and TCF and ECF bleached pulps is shown in Table 1.Flax fibers have the highest content on lipophilic extractives followed by hemp,sisal and abaca fibers. Regardless the higher lipid content of flax fibers, thecontent of lipophilic extractives of its crude pulp was similar or even lower thanin the other fibers. On the other hand, the lipid content of the pulps decreasedfurther after TCF or ECF bleaching, ranging from 0.03 to 0.18%. Thedifferences observed in the lipid content among the different crude and bleachedpulps of the nonwoody fibers is related to the behavior of lipophilic extractivespresent in the raw materials in alkaline pulping and to their reactivity towardsthe bleaching agents used as described below.3.1. Composition of lipophilic extractives in the nonwoody fibers selectedfor this studyWith the aim of studying the fate of the different lipophilic extractives duringsoda/AQ pulping, the lipid extracts from the different nonwoody fibers selectedfor this study were analyzed by GC and GC-MS and then compared with thosein the crude pulps. The composition of the lipophilic extractives of the selectedraw materials are listed in Table 2 and the chemical structures of the maincompounds identified are represented in Fig. 1.Table 1. Lipophilic extractives content (%) in the selected nonwoody raw materials andpulpsFlax Hemp Sisal AbacaFiber 1.70 0.88 0.68 0.51Crude pulp 0.21 0.26 0.30 0.20TCF pulp 0.05 0.18 0.09 0.04ECF pulp 0.13 0.18 0.14 0.03206

5. Resultados y discusiónIIIOHOOIIIHIVOHOHOVVIOCH 2 OHOHOOHOHOVIIHOVIIIOOIXFig.1. Chemical structures of compounds representing the main classes of lipophilicextractives found in lignocellulosic fibers: I, pentacosane; II, octacosanol; III, octacosanal;IV, palmitic acid; V, sitosterol; VI, sitosteryl linoleate; VII sitosteryl 3-D-glucopyranoside;VIII, -amyrin; and IX, octacosyl hexadecanoate.The main lipid classes present in the nonwoody fibers selected for this studywere 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 compoundspresent in these fibers have been previously addressed (del Río et al., 2004; delRío and Gutiérrez, 2006; Gutiérrez et al., 2006; 2008; Gutiérrez and del Río,2003a; 2003b).In the case of flax bast fibers the predominant lipophilic compounds presentwere series of fatty acids and aldehydes, accounting for 34% and 23% of totalextract, respectively, followed by ester waxes (18%), and fatty alcohols (13%).Fatty acids were also the predominant compounds (27% of total extracts) inhemp bast fibers, followed by alkanes (15%), free sterols (12%) and steroidhydrocarbons (12%). Among the selected leaf fibers, free sterols predominatedin both sisal (20%) and abaca (45%), followed by alkanes (14%) and fatty acids(10%) in sisal, and by fatty acids (16%) in abaca fibers.207

5. Resultados y discusiónThese lipophilic compounds can be classified into two principal groups,namely organic acids and neutral components. Organic acids include fatty acids,and the neutrals include alkanes, aldehydes, fatty alcohols, and free sterols aswell as esterified and conjugated compounds such as ester waxes, sterol esters,alkylferulates and sterol glycosides. Organic acids and neutral compounds, thelatter including saponifiable and unsaponifiable compounds present differentbehavior during alkaline pulping, as shown below.3.2. Fate of lipophilic extractives during soda/AQ pulpingThe fate of the different lipophilic extractives during soda/AQ pulping wasstudied by analyzing the respective crude pulps. This is exemplified in Figs. 2and 3 for the hemp bast fibers and the sisal leaf fibers, respectively. Thecomposition of main lipids present in the crude pulps from the studied fibers islisted in Table 2. It can be observed that the lipophilic compounds from the rawmaterials are modified to a different extent during alkaline (soda/AQ) pulpingdepending of their chemical nature. Fatty acids, which are among the mainlipophilic extractives predominating in all these fibers, were also present insignificant amounts in the crude pulps. The content of fatty acids decreasedsignificantly after soda/AQ pulping of flax fibers whereas an increase in fattyacids content was produced after pulping of the other fibers, being especiallyevident in the case of sisal and abaca. In contrast, -hydroxyfatty acids presentin all the fibers and -hydroxyfatty acids present in sisal and abaca werecompletely absent in crude pulps.On the other hand, esterified compounds such as ester waxes, sterols estersand ferulic acid esters, were completely hydrolyzed during soda/AQ pulping.This is especially evident in the case of flax fibers where ester waxespredominate. In contrast, other conjugated compounds, namely sterolglycosides, which are present in all nonwoody fibers studied here, resisted thealkaline cooking conditions and were present intact in the crude pulps, asalready reported in the pulping of woody (Gutiérrez and del Río, 2001;Nilvebrant and Byström, 1995) and nonwoody plants (Gutiérrez and del Río,2003a; 2003b; Gutiérrez et al., 2004). The importance of the presence of sterolglycosides after alkaline pulping is due to their high hydrophilic-lipophilicbalance, high melting point and very low solubility in water, alkali and the usualorganic solvents (Hillis and Sumimoto, 1989). Due to these properties, sterolglycosides constitute a part of protecting layers that prevent the cooking andbleaching chemicals to reach the resin and thereby keep them and otherextractives in the pulp. Other neutral compounds, such as aldehydes, which werepresent in significant amounts in flax and hemp fibers, decreased largely duringpulping and therefore were practically absent in the crude pulps. The content ofsteroid hydrocarbons and steroid ketones also decreased after soda/AQ cooking.208

5. Resultados y discusiónAk 29+5+6+711Hemp bast fibersAl 269 +10Ak 29Ak 31Fa 18:1+FaFa 18:2 16Ak 273 48+WAl 12 W 442442 W 46SE W48W 50Ak 31+Al 28Ak 29+Al 26ECF pulpAl 30115+6+7Fa 18:1Al 264 Al 30Ak 31+Al 28Crude pulpAQAk 2718:2+Al 24Fa 16 Fa 181+Fa311+5+6+7AQAk1127+ 1 34Al 24Ak 29+ +Al 26Al 28DefoamerTCF pulpFa 16 Fa18Al 30AQAl 22 Al +241Ak 277Fa 1812Fa 16Fa 18255 10 15 20Retention time (minutes)Fig. 2. GC-MS chromatograms of underivatized lipophilic extractives from hemp fibers (rawmaterial), 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 carbonatom number.209

5. Resultados y discusión5+6+7Sisal leaf fibersAl 281 4Ak 29+ 28Al 26Fa 16Ak F 24325Ak 21 Ak 27Ak 239Fe 24Fe 26 Fe 285+6+7Al 28Ak 29+Al 264AQAk 25Al 30Fa + +163 9Al 1 8Fa 22 Al 24 18Al 285+6Ak 29+Al 26DefoamerAk 254+Ak 27+Al 24AQ Fa 16 Fa 181 3Al 22Ak 27Al 309Crude pulpTCF pulpFa 16 ++AQAl 22145+6Ak 25Ak 27Fa 18Al 24Ak 29Al 28+Al 269ECF pulpAl 30255 10 15 20Retention time (minutes)Fig. 3. GC-MS chromatograms of underivatized lipophilic extractives from sisal fibers (rawmaterial), 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 thetotal carbon atom number.210

5. Resultados y discusiónTable 2. Composition of lipophilic extractives (mg/100 g) in nonwoody rawmaterials (M) and crude (C), TCF (T) and ECF (E) pulpsFlax Hemp Sisal AbacaCompound M C T E M C T E M C T E M C T EFatty acids 552 96 29 39 78 60 12 80 9 50 10 42 9 57 19 28n-hexadecanoic acid 121 27 4 18 34 15 4 29 4 24 8 27 4 20 9 109,12-octadecadienoic 1 tr - - 3 1 1 - - - - - 1 3 - -acid9-octadecenoic acid 235 39 2 1 15 1 tr 1 - - - - 2 6 - -n-octadecanoic acid 52 22 8 12 6 10 3 20

5. Resultados y discusiónAldehydes 371 3

5. Resultados y discusiónFinally, alkanes, fatty alcohols and free sterols and triterpenols survived thealkaline conditions and therefore were the main lipophilic constituents in crudepulps.The behavior of the fatty acids in an aqueous environment is quite differentfrom that of the neutrals. At alkaline pH, the acids dissociate and can dissolve inwater to quite a high extent forming fatty acid soaps that can form micelles.Fatty acid soaps are effective solubilizing agents facilitating the removal frompulp of sparingly soluble neutral substances (Back, 2000). The ratio ofsaponifiables-to-unsaponifiables has been suggested to be a better index forpredicting pitch problems than the total amount of lipids (Back and Allen,2000). In fact, the higher abundances of unsaponifiable compounds with respectto the saponifiable ones is the main cause for pitch problems during pulping ofsome woods, such as aspen or eucalypt (Chen et al., 1995; del Río et al., 1998;2000). This fact can explain why the lipid content of crude flax pulps is similaror even lower than the other pulps regardless flax fibers had the highestextractive content. As mentioned above, fatty acids are the predominat lipids inflax fibers and can help to solubilize other water-insoluble components such asfatty alcohols and sterols. In contrast, the lipid content of crude sisal pulp ishigher than the other three pulps regardless the relatively low content oflipophilic extractives in the raw material. Sisal fibers have low fatty acid contentwhereas the content in neutrals such as alkanes, fatty alcohols and steroids isrelatively high. Particularly, the high abundances of free sterols, which have ahigh propensity to form pitch deposits (del Río et al., 1998; 2000), in the crudepulps from hemp and sisal, would point to a high pitch deposition tendency ofthe lipophilics from these fibers.3.3. Fate of lipophilic extractives during TCF and ECF bleachingThe lipophilic extractives remaining in the crude pulp are carried over to thebleach plant, where they react with the bleaching agents used (Björklund-Jansson et al., 1995; Holmbom, 2000). Pulp bleaching technology radicallychanged in the 1990s when the previously used chlorine was replaced andseveral new bleaching chemicals were introduced (Sjöström, 1993) and ECF andTCF sequences were adopted. As a consequence, new pitch problems arose dueto the different reactivity of pulp lipids with the new bleaching agents. With theaim of studying the behavior of the different classes of lipophilic extractivesunder different bleaching conditions, both TCF and ECF bleached pulps wereanalyzed and compared with their respective crude pulps.In general terms, the qualitative composition of the lipophilic extractives fromthe TCF pulps was very similar to that of their respective crude pulps (Figs. 2and 3) although the lipid content was significantly lower (Table 2). Fattyalcohols, alkanes, free sterols and triterpenols, and fatty acids were the mainlipophilic compounds present in the TCF bleached pulps, although in loweramount than in their respective crude pulps. The low reactivity of most pulp213

5. Resultados y discusiónlipids in TCF bleaching sequences (using hydrogen peroxide as a bleachingagent) has been reported (Freire et al., 2003; Gutiérrez et al., 2001a). Thedecrease in the content of these lipophilic compounds in the TCF pulps,regardless the low reactivity of hydrogen peroxide towards them, might be dueto the extensive washing in alkaline conditions carried out in the industrial TCFbleaching sequences used. Moreover, it is interesting to note that sterolglycosides, which resisted soda/AQ pulping conditions, were removed to a highextent under the industrial TCF bleaching conditions used in this work. The highefficiency of hydrogen peroxide in degrading sterol glycosides was previouslyreported using model compounds (Nilvebrant and Byström, 1995). However, alower extent in the removal of sterol glycosides were observed during TCFbleaching of eucalypt pulps (Gutiérrez et al., 2006). In addition to the lipophilicextractives arising from the raw materials, several non-resolved peakscorresponding to the defoamers used in the process could also be observed inTCF pulps (Figs. 2 and 3). The excessive use of defoamers can also produceproblems of pitch deposition (Allen, 2000). The presence of defoamer in pitchdeposits produced during manufacturing of paper pulp from hemp fibers hasbeen reported (Gutiérrez and del Río, 2005).On the other hand, the composition of the lipophilic extractives from ECFpulps was somewhat different compared to that from their respective crudepulps (Figs. 2 and 3). The main difference observed was the large removal offree sterols in all the ECF pulps, although some minor amounts of free sterolsstill remained in sisal ECF pulp. The complete degradation of unsaturatedsterols, such as sitosterol, during ECF bleaching has been previously reported inpitch deposits, pulps and in reactions with pure compounds (Freire et al., 2003;Gutiérrez et al., 2001a; Björklund-Jansson et al., 1995). On the other hand,sterol glycosides were largely removed during the ECF bleaching and werepractically absent in these pulps. A less reactivity of these compounds withchlorine dioxide compared to hydrogen peroxide has been reported (Nilvebrantand Byström, 1995). Therefore, the complete removal of sterol glycosides in theECF pulps studied here may have been due to the use of hydrogen peroxide inthe ECF sequence.The removal of lipophilic extractives in bleaching is mainly achieved by twomechanisms: by dissolving or dispersing the pulp lipids followed by removalwith washing liquors, and by oxidation of lipids with bleaching chemicalsresulting in more hydrophilic compounds, which then may be dissolved inbleaching liquors and subsequently removed in washing (Holmbom, 2000). Thedecrease in the content of the lipophilic compounds in the TCF pulps studiedhere might be due to the first mechanism as mentioned above, taking intoaccount the low reactivity of these lipids towards hydrogen peroxide. Incontrast, the removal of unsaturated compounds during ECF bleaching was dueto the second mechanism (Holmbom, 2000). The lower reactivity of pulp lipidsin TCF bleaching sequences using hydrogen peroxide as a bleaching agent214

5. Resultados y discusióncompared to ECF bleaching using chlorine dioxide (Back and Allen, 2000;Gutiérrez et al., 2009) may cause pitch problems to be, in principle, more severein the former bleaching sequences. This is specially evident in the case ofunsaturated steroids and triterpenoids as well as unsaturated fatty acids, whichare strongly modified by chlorine dioxide but remain practically unaltered byoxygen and hydrogen peroxide (Holmbom, 2000; Bergelin and Holmbom, 2003;Freire et al., 2005; 2006; Gutiérrez et al., 2001a). However, in the nonwoodypulps studied here, the major lipophilic compounds present are saturated fattyacids, fatty alcohols, alkanes, which do not show reactivity towards chlorinedioxide, and therefore there are not great differences in the composition of thelipophilic extractives between ECF and TCF bleached pulps, with the exceptionof abaca pulp which lacks fatty alcohols and alkanes and where unsaturatedsterols are the predominant lipophilic compounds. Therefore, both ECF and TCFbleached pulps will undergo similar pitch problems. Fatty acids, fatty alcoholsand alkanes have been reported to be the compounds responsible for pitchdeposits formed during pulping of nonwoody plants (Gutiérrez and del Río,2005).4. ConclusionsA thorough chemical characterization of the lipophilic extractives from differentnonwoody fibers (flax, hemp, sisal and abaca) at different stages of pulpproduction (soda/AQ pulping and TCF/ECF bleaching) has been carried out.This study provides useful information into the extent of their removal along thecooking and bleaching processes. The soda/AQ pulping stage led to the removalof aldehydes, hydroxyfatty acids and the complete hydrolysis of esterifiedcompounds such as ester waxes, sterol esters and alkyl ferulates. Among thebleaching processes, ECF bleaching showed high reactivity towards unsaturatedsterols and both ECF and TCF bleaching were very effective in removing sterolglycosides from nonwoody pulps. In contrast, saturated fatty acids, fattyalcohols and alkanes, which are the main lipophilic compounds in most of thestudied fibers, survived pulping and bleaching conditions and were thepredominating compounds in both TCF and ECF bleached pulps.AcknowledgementsThis study has been supported by the Spanish projects BIO2007-28719-E andAGL2008-00709 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.215

5. Resultados y discusiónReferencesAllen, L.H., 2000. Pitch control in pulp mills, in: Back, E. L., Allen, L. H.(Eds.), Pitch control, wood resin and deresination. TAPPI Press, Atlanta, pp.265-288.Back, E.L., 2000. Deresination in pulping and washing, in: Back, E. L., Allen,L. H. (Eds.), Pitch Control, Wood Resin and Deresination. TAPPI Press,Atlanta, pp. 205-230.Back, E.L., Allen, L.H., 2000. Pitch control, wood resin and deresination.TAPPI Press, Atlanta.Bergelin, E., Holmbom, B., 2003. Deresination of birch kraft pulp in bleaching.J. Pulp Pap. Sci. 29, 29-34.Bergelin, E., Holmbom, B., 2008. Reactions and distribution of birch extractivesin kraft pulp oxygen delignification. J. Wood Chem. Technol. 28, 261-269.Bergelin, E., Möller, R., Holmbom, B., 2005. Analysis of pitch and depositsamples in kraft pulp production. Pap. Puu-Pap. Tim. 87, 399-403.Björklund-Jansson, M., Wormald, P., Dahlman, O., 1995. Reactions of woodextractives during ECF and TCF bleaching of kraft pulp. Pulp Pap. Can. 96,T134-T137.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 pitchproblems than softwoods in kraft pulping. Tappi J. 78, 143-149.del Río, J.C., Gutiérrez, A., 2006. Chemical composition of abaca (Musatextilis) leaf fibers used for manufacturing of high quality paper pulps. J. Agr.Food Chem. 54, 4600-4610.del Río, J.C., Gutiérrez, A., González-Vila, F.J., Martín, F., Romero, J., 1998.Characterization of organic deposits produced in the kraft pulping ofEucalyptus globulus wood. J. Chromatogr. A 823, 457-465.del Río, J.C., Rodríguez, I.M., Gutiérrez, A., 2004. Identification of intact longchainp-hydroxycinnamate esters in leaf fibers of abaca (Musa textilis) usinggas chromatography/mass spectrometry. Rapid Commun. Mass Sp. 18, 2691-2696.216

5. Resultados y discusióndel Río, J.C., Romero, J., Gutiérrez, A., 2000. Analysis of pitch depositsproduced 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., 2003. Oxidized derivatives oflipophilic extractives formed during hardwood kraft pulp bleaching.Holzforschung 57, 503-512.Freire, C.S.R., Silvestre, A.J.D., Neto, C.P., 2005. Lipophilic extractives inEucalyptus globulus kraft pulps. Behavior during ECF bleaching. J. WoodChem. Technol. 25, 67-80.Freire, C.S.R., Silvestre, A.J.D., Neto, C.P., Evtuguin, D.V., 2006. Effect ofoxygen, ozone and hydrogen peroxide bleaching stages on the contents andcomposition of extractives of Eucalyptus globulus kraft pulps. BioresourceTechnol. 97, 420-428.Gutiérrez, A., del Río, J.C., 2001. Gas chromatography-mass spectrometrydemonstration of steryl glycosides in eucalypt wood, kraft pulp and processliquids. Rapid Commun. Mass Sp. 15, 2515-2520.Gutiérrez, A., del Río, J.C., 2003a. Lipids from flax fibers and their fate inalkaline pulping. J. Agr. Food Chem. 51, 4965-4971.Gutiérrez, A., del Río, J.C., 2003b. Lipids from flax fibers and their fate inalkaline pulping (Vol 51, pg 4965, 2003). J. Agr. Food Chem. 51, 6911-6914.Gutiérrez, A., del Río, J.C., 2005. Chemical characterization of pitch depositsproduced in the manufacturing of high-quality paper pulps from hemp fibers.Bioresource Technol. 96, 1445-1450.Gutiérrez, A., del Río, J.C., González-Vila, F.J., Martín, F., 1998. Analysis oflipophilic extractives from wood and pitch deposits by solid-phase extractionand gas chromatography. J. Chromatogr. A 823, 449-455.Gutiérrez, A., del Río, J.C., Ibarra, D., Rencoret, J., Romero, J., Speranza, M.,Camarero, S., Martínez, M.J., Martínez, A.T., 2006. Enzymatic removal offree and conjugated sterols forming pitch deposits in environmentally soundbleaching of eucalypt paper pulp. Environ. Sci. Technol. 40, 3416-3422.Gutiérrez, A., del Río, J.C., Martínez, A.T., 2009. Microbial and enzymaticcontrol of pitch in the pulp and paper industry. Appl. Microbiol. Biot. 82,1005-1018.217

5. Resultados y discusiónGutiérrez, A., Rodríguez, I.M., del Río, J.C., 2004. Chemical characterization oflignin and lipid fractions in kenaf bast fibers used for manufacturing highqualitypapers. J. Agr. Food Chem. 52, 4764-4773.Gutiérrez, A., Rodríguez, I.M., del Río, J.C., 2006. Chemical characterization oflignin and lipid fractions in industrial hemp bast fibers used for manufacturinghigh-quality paper pulps. J. Agr. Food Chem. 54, 2138-2144.Gutiérrez, A., Rodríguez, I.M., del Río, J.C., 2008. Chemical composition oflipophilic extractives from sisal (Agave sisalana) fibers. Ind. Crop. Prod. 28,81-87.Gutiérrez, A., Romero, J., del Río, J.C., 2001a. Lipophilic extractives fromEucalyptus globulus pulp during kraft cooking followed by TCF and ECFbleaching. Holzforschung 55, 260-264.Gutiérrez, A., Romero, J., del Río, J.C., 2001b. Lipophilic extractives in processwaters during manufacturing of totally chlorine free kraft pulp from eucalyptwood. 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. TappiPress, Atlanta, pp. 231-244.Nilvebrant, N.-O., Byström, S., 1995. Demonstration of glucosidic linked sterolsin birch. Proc. 8th Intern. Symp. Wood and Pulping Chem., Helsinki, 6-9 June2, 135-140.Shin, S.J., Kim, C.H., 2006. Residual extractives in unbleached aspen and pinekraft 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 pitchdeposits from ECF Eucalyptus globulus bleached kraft pulp mill: Itsrelationship with wood extractives and additives in process streams. Appita J.52, 375-382.218

5. Resultados y discusiónSjöström, E., 1993. Wood chemistry. Fundamentals and applications. AcademicPress, San Diego.219

5. Resultados y discusiónPublicación VIII:Marques G., Gutiérrez A., del Río J.C. and Evtuguin D.V. (2010) Acetylatedheteroxylan from Agave sisalana and its behavior in alkaline pulping andTCF/ECF bleaching. Carbohydrate Polymers, 81, 517-523.220

5. Resultados y discusiónAcetylated heteroxylan from Agave sisalana and its behavior in alkalinepulping and TCF/ECF bleachingGisela Marques a , Ana Gutiérrez a , José C. del Río a and Dmitry V. Evtuguin ba Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, P.O. Box 1052, 41080-Seville, Spainb CICECO and University of Aveiro, Department of Chemistry,3810-193 Aveiro, PortugalAbstractThe heteroxylan from sisal (Agave sisalana), an O-acetyl-(4-Omethylglucurono)xylanwith a molecular weight (Mw) of 18 kDa, was isolatedby extraction of peracetic holocellulose with Me 2 SO and thoroughlycharacterized by wet chemistry, and NMR spectroscopy. The heteroxylanbackbone 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,

5. Resultados y discusión2008; Mwaikambo, 2006; Megiatto et al., 2007; Hurter, 2001; Idárraga et al.,2001). Easily bleachable sisal chemical pulp is industrially produced by sodapulping in the presence of athroquinone (AQ) as catalyst.The basic knowledge of the chemical composition of sisal fibers, as well asthe behavior of its components during pulping and bleaching, is essential for thebetter understanding and improving the pulping and bleaching operations andfor the assessment of pulp and paper properties. Previous papers have reportedthe composition of the lipophilic compounds (Gutiérrez et al., 2008) and thestructure of the lignin (del Río et al., 2007, 2008) of sisal fibers, but only limitedwork has been performed on the structural characterization of the carbohydratefraction of this fiber (Das Gupta & Mukherjee, 1967; Stewart et al., 1997;Megiatto et al., 2007). In the present work, we report the structuralcharacterization of a heteroxylan in sisal fibers, as well as in their soda/AQpulps (unbleached and TCF/ECF bleached). The study of hemicelluloses is offundamental and practical interest, since their partial degradation and dissolutionduring 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 boundedto cellulose fibrils. Hemicelluloses are mainly branched polymers of lowmolecular 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; Ebringerová et al., 2005). In particular, glucuronoxylan (GX) isthe major hemicellulose in such important industrial crops produced by agroindustryas kenaf, bamboo, flax, sisal and jute and is structurally similar tohardwood 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/compositionalstudies of GX from sisal were carried out mainly with alkali-extracted GX(Stewart et al., 1997; Das Gupta & Mukherjee, 1967). Hence, some essentialstructural information, such as substitution patterns with acetyl groups, was notassessed. It was suggested, however, that sisal GX is an O-acetyl-4-Omethylglucuronoxylanwith moderate molecular weight, around 15-20 kDa(Stewart et al., 1997; Das Gupta & Mukherjee, 1967). According to data frommethylation analysis of sisal xylan, its backbone is constituted by -(1-4)-linkedD-xylopyranose residues, carrying a low degree of substitution (8-10 % mol.)with terminal 4-O-methyl-D-glucopyranosyluronic acid residue linked throughthe O-2 position (Das Gupta & Mukherjee, 1967).In this report, the heteroxylan from sisal fibers was isolated by extraction ofperacetic holocellulose with dimethyl sulfoxide and thoroughly characterized bywet chemistry and NMR spectroscopy. This isolation procedure allowed to222

5. Resultados y discusiónobtain a xylan sample with intact O-acetyl moieties. Simultaneously, the fate ofthis heteroxylan structure during soda/anthraquinone pulping and TCF and ECFbleaching was also studied.2. Experimental2.1. SamplesSisal (Agave sisalana) leaf fibers from Africa (Kenya), their soda/AQ chemicalpulps (unbleached and ECF/TCF bleached) and cooking black liquor weresupplied by CELESA pulp mill (Tortosa, Spain). The TCF bleaching sequenceE(O)P-EP included two hydrogen peroxide stages at 90ºC, the first underoxygen pressure (E(O)P stage) and the second without oxygen (EP stage). TheECF bleaching sequence D-EP included a chlorine dioxide (D) at 60ºC and ahydrogen peroxide (EP) stages at 90ºC.The samples were air-dried, milled using a knife mill (Janke and Kunkel,Analysenmühle), and extracted with acetone in a Soxhlet apparatus for 8 h. Forestimation of the Klason lignin content, the acetone extracted samples weresubsequently extracted with hot water (3 h at 100 ºC). Klason lignin wasestimated as the residue after 72% sulfuric acid hydrolysis of the pre-extractedmaterial according to Tappi standard procedures (Tappi Test Methods, 1993).Ash content was estimated as the residue after 6 h calcination at 575 ºC.2.2. Preparation of holocelluloseThe holocelluloses of sisal fibers and their unbleached pulp were obtained fromextractives-free sawdust (5.0 g) by delignification with 10% peracetic acid at pH3.5 for 20 min at 85 ºC. After delignification, the holocellulose was filtered offon a porous glass filter, washed with acetone and further with warm water andair-dried. The holocellulose yield was 69.7% and 94.7% for the sisal fibers andunbleached pulp, respectively.2.3. Isolation of xylans from pulpsThe isolation of acidic heteroxylans was carried out by two consecutiveextractions with Me 2 SO (1.5 g of holocelluloses with 50 mL of Me 2 SO in eachassay) at 50 ºC for 24 h under stirring and further precipitation of the resultedextracts in an excess of 7:2:1 EtOH-MeOH-water acidified by HCOOH. Thecomplete precipitation of the heteroxylan was accomplished in 3 days at 4 ºC.The heteroxylan was isolated by centrifugation, washed four times withanhydrous MeOH and quickly dried under vacuum at room temperature. For theECF and TCF bleached pulps the xylans were extracted directly from the pulpwithout previous preparation of holocelluloses.223

5. Resultados y discusión2.4. Isolation of xylan from the black liquorThe xylan of the black liquor was isolated following a procedure previouslydescribed, with minor modifications (Engström et al, 1995). 200 ml of 1,4-dioxane were slowly added with agitation to 100 ml of diluted with distilledwater 1/2 black liquor, followed by the addition of glacial acetic acid until pH 2-3. The solution and resulting precipitate was kept at 5 ºC during 2 days. Theblack liquor precipitated polysaccharides (BLPS) were separated bycentrifugation and the solution decanted off. The precipitate was sequentiallywashed up with 150 ml of a 1,4-dioxane-water (2:1) solution, 150 ml of 1,4-dioxane, 150 ml of methanol and 150 ml of acetone and, finally, dried undervacuum with phosphorus pentoxide.2.5. Carbohydrate analysisThe heteroxylan was subjected to hydrolysis with 72% H 2 SO 4 at 20 ºC for 3 h,followed by 2.5 h hydrolysis with diluted 1 M H 2 SO 4 at 100 ºC (Saemanhydrolysis) and the released neutral monosaccharides were determined as alditolacetate derivatives by gas chromatography (Selvendran et al., 1979). Thequantitative analysis was carried out on a Varian 3350 gas chromatographequipped with a FID detector and with a DB-225 J&W column (30 m×0.25 mmi.d. ×0.15 m film thickness). The temperature program was started at 220 ºCwith a 5 min hold, and then raised to a final temperature of 230 ºC at 2 ºC/min,and held for 5 min. The injector and detector temperatures were set at 230 ºC.The quantification was made using calibration curves with standards.2.6. Acid methanolysis for analysis of sugars and uronic acidsAbout 4.5 mg of freeze-dried sisal fibers and their pulps (unbleached, ECF andTCF bleached pulps) were subjected to acid methanolysis by the addition of 2mL, 2 M solution of HCl in anhydrous methanol at 100 ºC for 4 hours(Sundberg et al., 1996). After cooling to room temperature, about 80 L ofpyridine was added to neutralize the acidic solution. Additionally, 1 mL ofinternal standard solution containing 0.1 mg/mL of sorbitol was added. To avoidfibers silylation, 2 mL of the supernatant reaction solution was separated fromfiber suspension and evaporated in a rotary evaporator with a water bath kept at40-50 ºC. The samples were dissolved by addition of 70 L pyridine. Forsilylation, 150 L hexamethyldisiloxane and 80 L trimethylchlorosilane wereadded and the samples were shaken well. After 12 hours at room temperature,the samples were ready for analysis. GC-MS analysis were performed using aHewlet-Packard Gas Chromatograph 5890 equipped with a mass selectivedetector MSD series II, using helium as carrier gas (35 cm/s), equipped with aDB-1 J&W capillary column (30 m×0.32 mm i.d. 0.25 m film thickness). Thecolumn temperature program was 100 – 4 ºC/min – 175 ºC followed by 175 – 12ºC/min – 290 ºC. The detector (FID) temperature was 290 ºC. The different224

5. Resultados y discusiónpeaks were identified by comparing their mass spectra with mass spectra inWiley and NIST libraries and that reported in the literature (Sundberg et al.,1996; Bertaud et al., 2002; Bleton et al., 1996).2.7. Size-exclusion chromatography (SEC)The xylan samples were dissolved in a small amount of 10% LiCL solution inN,N-dimethylacetamide (DMAC) at 70-80 ºC and further diluted with DMAC toa xylan concentration of about 0.5% (5mg/mL). The SEC analysis has beencarried out on two PLgel 10 μm MIXED B 300 × 7.5 mm columns protected bya PLgel 10 μm pre-column (Polymer Laboratories, UK) using a PL-GPC 110system (Polymer Laboratories). The columns, injector system and the detector(RI) were maintained at 70 ºC during the analysis. The eluent (0.1 M LiClsolution in DMAC) was pumped at a flow rate of 0.9 mL/min. The analyticalcolumns were calibrated with pollulan standards (Polymer Laboratories) in therange 0.8-100 kDa. The injected volume was 100 μL.2.8. NMR spectroscopyOne-dimensional 1 H NMR spectra of the xylan samples were recorded in D 2 O(30 ºC) on a Bruker Avance 300 spectrometer operating at 300.13 MHz. Sodium3-(trimethylsilyl)-propionate-d 4 was used as internal standard ( 0.00). Therelaxation delay was 16 s, r.f. 90º-pulse width of 10.2 μs and about 400 pulseswere collected. All 2D NMR spectra were recorded on a Bruker Avance 300spectrometer operating at 300.1 MHz for proton and at 75.2 MHz for carbon.2D 1 H– 1 H COSY spectroscopy was performed at 50 °C using a standard COSYsequence (90° pulse, relaxation delay 2 s). Two-dimensional 1 H- 1 H TOCSY(Total Correlation Spectroscopy) spectra (mix= 0.050 s) were acquired at aspectral width of 2185 Hz in both dimensions at 60 ºC. The relaxation delay was2.0 s. For each FID, 128 transients were acquired; the data size was 1024 in t 1 ×512 in t 2 . The phase sensitive 1 H-detected HSQC (Heteronuclear SingleQuantum Coherence) spectrum was acquired at 50ºC over a F1 spectral weightof 12,000 Hz and a F2 width of 2000 Hz with a 2048 × 1024 matrix and 128transients per increment. The delay between scans was 2 s and the delay forpolarization transfer was optimized for 1 J CH = 148 Hz.2.9. Hexenuronic acid contentThe amount of hexenuronic acids (HexA) was determined by acidic hydrolysisin sodium formate buffer at pH 3.0 followed by UV detection of furanderivatives at 245 nm (Vuorinen et al. 1999).225

5. Resultados y discusión3. Results and discussion3.1. Chemical composition of sisal fibersThe chemical composition of sisal (Agave sisalana) fibers is presented in Table1. The sugar analysis confirmed the data previously reported by Stewart et al.(1997) indicating that xylan is the principal hemicellulose of sisal fibers and thesecond most abundant polysaccharide after cellulose. At the same time, takinginto account the small amount of lignin (around 6%) and extractives (slightlymore than 3%) in sisal fibers, the relatively low yield of peracetic holocellulose(70%) indicates a significant content of easily removable hemicelluloses, otherthan xylan. These may be pectins and, in particular, glucans that are known to bepresent in the leaves of the genus Agave in noticeable amounts (Nobel, 2003).The misbalance in cellulose content and the amount of glucose detected uponsugars analysis (Table 1) allows suggesting that non-cellulosic glucans maycontribute to at least 15% (w/w) of sisal fibers. This fact was further confirmedby analysis of the hemicelluloses dissolved in the black liquor from soda pulpingof sisal fibers.3.2. Isolation and structural characterization of xylan from sisal fibersThe heteroxylan (yield of about 60% w/w) from sisal fibers was isolated fromperacetic holocellulose by two consecutive extractions with Me 2 SO followed byprecipitation of the extracted polyose in 7:2:1 ethanol/methanol/water. Suchprocedure guaranteed the isolation of intact and representative xylan sample,which can be structurally characterized including the quantification anddistribution of O-acetyl moieties (Evtuguin et al. 2003).Table 1. Chemical composition of sisal fibers, unbleached soda pulp and ECF and TCFbleached pulps (% w/w).Component Sisal fibers Unbleached pulp TCF pulp ECF pulpAsh 1.0 1.0 0.4 0.4Extractives (acetone) 0.8 0.3 0.1 0.1Extractives (water) 2.3 0.7 0.6 0.4Klason lignin 5.9 0.7 - -Holocellulose 70.0 95.0 - -Cellulose* 54.5 - - -Neutral sugarsRha 0.7 0.7 tr trAra 1.3 tr tr trXyl 20.0 19.0 19.4 20.6Man 0.8 - - -Gal 1.0 tr tr trFuc

5. Resultados y discusiónThe composition of the isolated xylan was assessed by analyses of neutralsugars and easily hydrolyzed sugars after methanolysis (Tables 2 and 3). Thehigh purity of the isolated xylan was confirmed using neutral monosaccharidesanalysis, that showed the predominance of xylose (Xyl) and only small amountsof glucose (Glc), galactose (Gal), arabinose (Ara) and rhamnose (Rha) (Table2). The presence of Gal, Ara and Rham may indicate the eventual smallcontamination of xylan with pectin compounds. This was confirmed bymethanolysis studies (Table 3, Figure 1), which revealed a much higher amountof galacturonic acid than could be expected if arisen only from the terminalstructural fragment [3)--L-Rhap-(12)--D-GalpA-(14)-D-Xylp]suggested to be present in xylans (Shimizu, 1991). The presence of D-glucopyranosyluronic acid (GlcpA), besides the expected 4-O-methyl-Dglucopyranosyluronicacid (MeGlcpA), may indicate that, at least part ofglucuronosyl moieties attached to xylan backbone, is not methylated. The ratiobetween internal xylopyranosyl units (Xylp) in the backbone and terminalattached glucuronosyl residues (MeGlcpA and GlcpA) was estimated to bearound 9:1. The molecular weight (M w ) of sisal xylan was about 18 kDa, asrevealed by SEC analysis (Figure 2).Table 2. Neutral monosaccharide composition (% w/w) of xylans isolated from sisal fibers,pulps and black liquor.Rha Ara Xyl Man Fuc Gal GlcSisal fibers 0.9 0.7 93.7 tr - 1.2 3.5Unbleached pulp tr tr 99.5 - - tr 0.5TCF bleached pulp tr tr 99.0 - - tr 1.0ECF bleached pulp tr tr 98.7 - - tr 1.3Black liquor precipitated polysaccharides(BLPS) 0.7 3.6 10.9 2.6 1.1 27.1 54.0tr: tracesAccording to previously reported methylation analysis of alkali extractedsisal xylan, its backbone is constituted by -(14)-linked D-xylopyranoseresidues branched at O-2 with terminal 4-O-methyl-D-glucopyranosyluronicacid residues (Das Gupta & Mukherjee, 1967). These structural features wereconfirmed by 1D and 2D NMR techniques. Single (COSY) and multiple(TOCSY) bonds 1 H- 1 H correlation analyses and 1 H- 13 C (HSQC) correlations(Figure 3) allowed assignment of proton and carbon signals in sisal heteroxylan,as summarized in Table 4. Chemical shifts of protons and carbons werepractically the same as those reported for acetylated heteroxylans from otherplant sources (Teleman et al. 2002; Evtuguin et al. 2003). The anomeric regionin the TOCSY spectrum (Figure 4) revealed the characteristic protoncorrelations that are normally found in heteroxylans containing attached tobackbone non-methylated GlcpA residues (Vignon & Gey, 1997; Gonçalves etal. 2008). Hence, NMR results corroborates the data obtained by methanolysis227

5. Resultados y discusiónTable 3. Neutral monosaccharide and uronic acids composition (% w/w) determined bymethanolysis of xylans isolated from sisal fibers and their pulps.Rha Ara Xyl Man Gal Glc GalA GlcA MeGlcpASisal fibers 0.8 0.6 83.0 - 0.8 1.3 2.8 0.3 10.5Unbleached pulp - - 98.3 - - 0.2 - - 1.5TCF bleached pulp - - 98.0 - - 0.8 - - 1.2ECF bleached pulp - - 97.5 - - 1.2 - - 1.3(Table 3), evidencing that a small proportion of glucuronic residues in sisalheteroxylan are not methylated. Therefore, it can be suggested that the backboneof sisal heteroxylan is composed of partially acetylated (14)-linked -D-Xylpunits O-2 ramified with terminal (12)-linked MeGlcpA and GlcpA.AraAraAraXylRhaXyl4-O-MeGLCAXylGalAXylGalAGalGalGalAGalAGalGlcGlc GlcAISSISAL FIBERSDimersSISAL UNBLEACHED PULPSISAL TCF PULPSISAL ECF PULP8 10 12 14 16 18 20 22 24 26Retention Time (min)Figure 1. Gas chromatogram of methylated and silylated sugars obtained by acidmethanolysis of xylans isolated from sisal fibers and their pulps. Xyl: xylose, Gal: galactose,Glc: Glucose, Rha: rhamnose, GlcA: glucuronic acid, 4-O-MeGlcA: 4-O-methylglucuronicacid, GalA: galacturonic acid.228

5. Resultados y discusiónTable 4. Proton/carbon chemical shifts () of heteroxylan from sisal (30ºC, D 2 O).Structural unitAssignmentsH1/C1 H2/C2 H3/C3 H4/C4 H5/C5axeqXyl (isol) 4.47/102.9 3.28/73.7 3.55/74.5 3.80/77.4 3.40/63.9 4.10/63.9Xyl-3Ac 4.57/102.4 3.49/71.9 4.98/76.3 3.93/76.5 3.47/63.8 n.d/63.8Xyl-2Ac 4.68/101.2 4.68/74.6 3.80/72.5 3.87/77.2 3.45/63.8 n.d/63.8Xyl-2,3Ac 4.80/100.5 4.82/74.6 5.16/74.0 4.06/76.6 3.54/63.9 n.d/63.9Xyl-3Ac-2GlcA 4.73/102.1 3.70/75.8 5.06/75.8 3.98/77.2 3.50/n.d n.d/n.dMeGlcA 5.27/98.9 3.57/72.3 3.88/73.7 3.27/83.2 n.d/n.d _ aGlcA 5.25/n.d 3.60/n.d 3.82/n.d 3.52/n.d n.d/n.d _ an.d non determined, a not relevant. The following designations were used: Xyl (isol.), non-acetylated Xylp in thebackbone isolated from other acetylated Xylp units; Xyl-3Ac, 3-O-acetylated Xylp; Xyl-2Ac, 2-O-acetylatedXylp; Xyl-2,3Ac, 2,3-di-O-acetylated Xylp; Xyl-3Ac-2GlcA, MeGlcA 2-O-linked and 3-O-acetylated Xylp;MeGlcA, 2-O-linked MeGlcpA; GlcA, 2-O-linked GlcpA.The acetylation patterns in the heteroxylan backbone were assessed by 1 HNMR spectroscopy based on signal assignment employing 2D NMR techniques(Table 4). The total 1 H NMR spectrum of sisal xylan and its expanded anomericregion with specified groups of protons in particular substructures, are presentedin Figure 5. According to previously published methodology (Evtuguin et al.2003), internal non-acetylated, 3-O- and 2-O-acetylated xylose residues,MeGlcpA residues were assessed based on their anomeric proton resonances,whereas the amounts of 2,3-di-O-acetylated and 3-O-acetylated/ MeGlcpA O-2substituted internal xylose residues were estimated based on H-3 resonances incorresponding structures (Figure 5). This allowed the integration of protonsfrom particular structural fragments and their quantification (Table 5). Around61%mol. of the Xylp residues were acetylated; among them, 39 %mol.corresponded to 3-O-acetylated, 13% mol. corresponded to 2-O-acetylated and1200960Response72048014 kDa12 kDa(D)(C)24010 kDa18 kDa(B)0(A)11.2 12.3 13.5 14.6 15.7Retention time (min)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 discusión9 %mol. corresponded to 2,3-di-O-acetylated residues. Accordingly, sisalheteroxylan possessed a substitution degree with acetyl groups of 0.70. Worthnotably, Xylp units in backbone of sisal heteroxylan are predominantly 3-Oacetylated.The proportion of 3-O-acetylated Xylp units in backbone was muchhigher (almost twice) in sisal xylan than in xylans from woody sources such asbirch and beech (Teleman et al. 2002), eucalypt (Evtuguin et al. 2003),paulownia (Gonçalves et al. 2008) and aspen (Teleman et al. 2000). Almost allXylp linked at O-2 with MeGlcpA (9 mol%) were 3-O-acetylated (Table 5).3.3. Changes in xylan structure during alkaline pulpingDuring the soda/AQ pulping, around 40% of the xylan was dissolved in theblack liquor. This conclusion was made based on the xylose content indelignified unbleached sisal fibers (Table 1) and the pulp yields (ca. 60%). Thechemical changes in the xylan during pulping were examined comparing thecomposition and structural features of the xylan from sisal fibers and their sodapulp, both isolated by Me 2 SO extraction of the corresponding peraceticholocelluloses (Tables 2 and 3, Figure 6).The molecular weight of the xylan remaining in the pulp decreased to 10 kDa,reflecting significant alkali-induced depolymerization (Figure 2). The xylansuffered also a significant deacetylation (about 95%) and the major part of theuronic moieties (at least 75%) were converted to 4-deoxy--L-threo-hex-4-enopyranosyluronic acid (hexenuronic acid or HexA), as revealed from 1 H NMRX5Ac3MeGlcAX5’MeGlcA3 X3Ac2X2Ac2X3Ac3MeGlcAX4Ac3MeGlcAX3Ac3X4Ac2 X4-OCH 3in MeGlcAMeGlcA2X3X5X2Ac3X2607080X1Ac2MeGlcA1X1Ac3X1Ac3MeGlcAX1X2Ac3MeGlcAMeGlcA4901005.5 5.0 4.5 4.0 3.5ppmFigure 3. HSQC spectrum (D 2 O, 50 ºC) of heteroxylan from sisal fibers.230

5. Resultados y discusiónTable 5. Relative content in acetyl groups in structural units of sisal heteroxylan.Relative abundanceStructural fragment and short designation(per 100 Xylp units)4)--D-Xylp-(1 (Xyl) 394)[3-O-Ac]--D-Xylp-(1 (Xyl-3Ac) 304)[2-O-Ac]--D-Xylp-(1 (Xyl-2Ac) 134)[3-O-Ac][2-O-Ac]--D-Xylp-(1 (Xyl-2,3Ac) 94)[4-O-Me--D-GlcpA-(12)][3-O-Ac]--D-Xylp--(1 (Xyl-3Ac-2MeGlcA) 94-O-Me--D-GlcpA-(1 (MeGlcA) 9spectra (Figure 6). The presence of HexA, formed under alkaline pulpingconditions via -elimination of methoxyl group, was confirmed applying thetotal correlation spectroscopy (TOCSY), according to previously publishedproton chemical shifts (Teleman et al. 1995). The HexA residues may be easilydetected in the anomeric region of 1 H NMR spectra, which showed theappearance of new signals at 5.36 and at 5.82 ppm that were assigned to H-1 andH-4 in corresponding structures (Figure 6). The HexA content in sisal soda pulpIIIIIV’ V IVIIH1/H4H1/H2H1/H3H1/H4H1/H2H3/H2H3/H5H3/H43.54.0H3/H1H3/H1H3/H1H14.5H1H3H3H3H1/H35.05.55.4 5.3 5.2 5.1 5.0 4.9 4.85.5ppmI- MeGlcpA-(1II- GlcpA-(1IV, IV’- 4)[3-O-Ac][2-O-Ac]--D-Xylp-(1V- 4)[4-O-Me--D-GlcpA-(12][3-O-Ac]--D-Xylp-(1III- 4)[3-O-Ac]--D-Xylp-(1Figure 4. Anomeric region of the TOCSY spectrum (D 2 O, 60 ºC) of heteroxylan from sisalfibers231

5. Resultados y discusiónwas 60.6 mmol/kg of pulp as determined after pulp treatment under acidicconditions followed by detection of furoic acid derivatives by UV-spectroscopyat 245 nm (Vuorinen et al. 1999).The balance of uronic moieties in the initial xylan and in the xylan remainingin the pulp was estimated based on the ratio of anomeric protons in uronicgroups 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 about30% of all uronic units (MeGlcpA and HexA) from xylan during pulping.The polysaccharides dissolved in the black liquor (BLPS) during pulpingwere isolated according to a previously published procedure (Engström et al,1995) and chemically characterized (Table 2). The aim of this study was tocompare the structure of the xylan remaining in the pulp with that dissolved inthe pulping liquor. Surprisingly, the heteroxylan was a minor polysaccharidedissolved in the liquor and its purification by fractional precipitation failed. Atthe same time, the analysis of neutral sugars of BLPS revealed glucans as themajor precipitated polysaccharides (glucose represents around 54 % of BLPSweight) followed by galactans. The preliminary study on BLPS using multiplebonds 1 H- 1 H correlation NMR spectroscopy (TOCSY) gave additional insightsH 2 O*CH 3 -CO -*5.5 5.0 4.5 4.0 3.5 3.0 2.5 ppmH1 H3 Xyl-MeGlcA 3Ac-2GlcAH3Xyl-2,3AcH3 Xyl-3AcH1/H2Xyl-2AcH1Xyl-3AcH1Xyl5.55.4 5.3 5.2 5.1 5.0 4.94.8 4.7 4.6 4.5 4.4ppmFigure 5. 1 H NMR spectrum (D 2 O, 30 ºC) of heteroxylans from sisal fibers (top image) andthe expanded region of anomeric protons (bottom image). The designations for the structuralfragments are the same as in Table 5. * solvent impurities.232

5. Resultados y discusiónSISAL UNBLEACHED PULPH1 XylH1MeGlcAH4 HexAH1HexASISAL TCF PULPSISAL ECF PULP5.9 5.7 5.5 5.3 5.1 4.9 4.7 4.5 4.3ppmFigure 6. 1 H NMR spectra (D 2 O, 30 ºC ) of heteroxylans isolated from sisal unbleached pulpand 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 -glucans, in particular, -(13)-glucans with a low degree of ramification at C6 (callose type), by comparison ofthe proton-proton correlations with previously published data on protonresonances in -(13)-glucan (Torosantucci et al. 2005). However, a moredetailed study is required to elucidate the exact structure of the -glucans in sisalfibers.3.4. Changes in xylan structure during TCF and ECF bleachingThe structural changes in the heteroxylan from sisal soda pulp during industrialTCF and ECF bleaching were also investigated. The chemical composition andstructural features were assessed in xylan samples isolated directly frombleached pulps by Me 2 SO extraction. The TCF pulp was obtained by E(O)P-EPbleaching and the ECF pulp was obtained by D-EP sequence. TCF pulp wasbleached essentially by hydrogen peroxide under alkaline conditions andincluded two hydrogen peroxide stages, the first with oxygen and the secondwithout oxygen, at 90 ºC,, whereas ECF pulp bleaching included a treatmentwith chlorine dioxide (D) at 60ºC followed by hydrogen peroxide stage underalkaline conditions (EP) at 90 ºC.233

5. Resultados y discusiónOHOHHHHOHOHOHOOOOOHOHHHHHH6axH5H1 H3 H2 H43.504.004.50ppm (t2)4.504.003.50ppm (t1)Figure 7. TOCSY spectrum (D 2 O, 60 ºC) of BLPS fraction isolated from black liquor. Topimage represents the fragment of -(1-3)-D-glucan backbone.The chemical analysis of the sugars of TCF and ECF bleached pulps did notshow significant changes when compared to the unbleached pulp (Table 1). Thisindicates that no specific removal of xylan from pulp took place duringbleaching. The chemical composition of the xylans isolated from TCF and ECFbleached pulps was also similar to the xylan from unbleached pulp, although avery small decrease in MeGlcpA content in pulp xylans after bleaching wasdetected (Tables 2 and 3). This fact may be explained by a partial degradationof MeGlcpA to HexA under alkaline conditions, which are inaccessible for theanalysis by methanolysis. This explanation was further supported by 1 H NMRanalysis, that showed a relative increase of the HexA content and a decrease ofMeGlcpA moieties in the xylan from TCF bleached pulp (Figure 6). In contrastto the xylan from TCF bleached pulp, the xylan from ECF bleached pulp did notcontain HexA residues, which were degraded upon bleaching with chlorinedioxide (Figure 6). Taking into account that uronic moieties strongly affect thepapermaking properties (Lindström, 1992) and brightness stability (Buchert atal. 1997) of cellulosic pulps this knowledge may be important to explain thedifferent response of pulps bleached employing TCF and ECF sequences.234

5. Resultados y discusiónThe xylans from bleached pulps (either TCF or ECF) did not show any acetylgroups, as revealed by 1 H NMR analysis. Hence, alkaline bleaching stagesfavored the removal of residual acetyl groups from xylan of unbleached pulp.Xylans from bleached pulps possessed slightly higher molecular weight (12 kDain TCF pulp and 14 kDa in ECF pulp), when compared to this in unbleachedpulp (Figure 2). This fact may be explained by a predominant removal of lowmolecular weight xylan fractions structurally associated to residual lignin duringbleaching.4. ConclusionsThe structure of the heteroxylan isolated from sisal fibers has been characterizedand its behavior during soda/AQ pulping and TCF/ECF bleaching has beenstudied. 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 smallproportion of -D-glucuronosyl (GlcpA) residues. Around 61mol% of the Xylpresidues are acetylated, the major proportion of acetyl groups being attached atthe O-3 position of the Xylp residues (39 %mol.), followed by acetylation at theO-2 position (13 %mol.) and diacetylation at both O-2 and O-3 positions(9%mol.). The molecular weight (M w ) of the heteroxylan was of 18 kDa.Around 40% of xylan was removed during soda pulping. However, the majorpolysaccharides found in the black liquor were -glucans rather than xylans.Sisal xylan suffered a significant depolymerisation (M w decreased to almosthalf) and deacetylation (95%) during pulping. Terminal MeGlcpA residues werepartially removed (to about 30%) or converted to HexA in a large extent. HexArevealed to be relatively stable during TCF bleaching with hydrogen peroxideand were predominant among uronic moieties of xylan. Since all HexA weredegraded during ECF bleaching with chlorine dioxide, the final pulp contained axylan with rather small amount of uronosyls (MeGlcpA residues). A smallproportion of MeGlcpA residues (15% from initial amounts), remaining intactduring soda pulping, were additionally converted to HexA residues during alkalibleaching stages. After bleaching, the residual acetyl groups were completelyremoved from the pulp xylan. It was suggested that a low molecular weightfraction of xylan, probably associated to residual lignin, was removed from uponbleaching.AcknowledgementsThis study has been supported by the Spanish Projects AGL2005-01748 andAGL2008-00709 and the EU BIORENEW project (NMP2-CT-2006-26456).We thank CELESA (Tortosa, Spain) for providing the samples. G.M. thanks theSpanish Ministry of Education for a FPI fellowship235

5. Resultados y discusiónReferencesBertaud, F., Sundberg, A., & Holmbom, B. (2002). Evaluation of acidmethanolysis for analysis of wood hemicelluloses and pectins. CarbohydratePolymers, 48, 319-324.Bleton, J., Mejanelle, P., Sansoulet, J., Goursaud, S., & Tchapla, A. (1996).Characterization of neutral sugars and uronic acids after methanolysis andtrimethylsilylation for recognition of plant gums. Journal of ChromatographyA, 720, 27-49.Buchert, J., Bergnor, E., Lindblad, G., Viikari, L., & Ek, M. (1997).Significance of xylan and glucomannan in the brightness reversion of kraftpulps. Tappi Journal, 80(6), 165-170.Das Gupta P. C., & Mukherjee P. P. (1967) The hemicellulose of sisal fibre(Agave sisalana). Journal of the Chemical Society C: Organic, 1179-1182.del Río, J. C., Marques, G., Rencoret, J., Martínez, A. T., & Gutiérrez, A.(2007). Occurrence of naturally acetylated lignin units. Journal of Agricultureand Food Chemistry, 55, 5461-5468.del Río, J. C., Rencoret, J., Marques, G., Gutiérrez, A., Ibarra, D., Santos, J. I.,Jiménez-Barbero, J., & Martínez, A. T., (2008) Highly acylated (acetylatedand/or p-coumaroylated) native lignins from diverse herbaceous plants.Journal of Agriculture and Food Chemistry, 56, 9525-9534.Ebringerová, A., Hromádková, Z., & Heinze, T. (2005). Polysaccharides I.Advances in. Polymer Science, 186, 1-67.Engström, N., Vikkula, A., Teleman A., & Vuorinen, T. (1995). Structure ofhemicellulose in pine kraft cooking liquors. Proceedings of the eighthInternational Symposium on Wood and Pulping Chemistry, (pp. 195-200), 6-9June, Helsinki, Finland.Evtuguin, D. V., Tomás, J. L., Silva, A. M. S., & Pascoal Neto, C. (2003).Characterization of an acetylated heteroxylan from Eucalyptus globulus labill.Carbohydrate Research, 338, 597-604.Gonçalves, V., Evtuguin D. V., & Domingues, M. R. (2008). Structuralcharacterization of acetylated heteroxylan from the natural hybrid Paulowniaelongate/Paulownia fortunei. Carbohydrate Research, 343, 256-266.Gorshkova, T. A., Wyat, S. E., Salnikov, V. V., Gibeaut, D. M., Ibragimov, M.R., Lozovaya, V. V., & Carpita, N.C. (1996). Cell-wall polysaccharides ofdeveloping flax plants. Plant Physiology, 110, 721-729.236

5. Resultados y discusiónGutiérrez, A., Rodríguez, I. M., del Río, J. C. (2008). Chemical composition oflipophilic extractives from sisal (Agave sisalana) fibers. Industrial Crops &Products, 28, 81-87.Hurter, R. W. (2001). Sisal fibre: market opportunities in the pulp & paperindustry. FAO/CFC Technical Paper, 14, 61-74.Idárraga, G., Ramos, J., Young, R. A., Denes, F., & Zuñiga, V. (2001).Biomechanical pulping of Agave sisalana. Holzforschung, 55, 42-46.Li, Y., Mai, Y.-W., & Ye, L. (2000). Sisal fibre and its composites: a review ofrecent developments. Composites Science and Technology, 60, 2037-2055.Lindström, T. (1992). Chemical factors affecting the behaviour of fibres duringpapermaking. Nordic Pulp and Paper Research Journal, 4(7), 181-192.Lisboa, S. A., Evtuguin, D. V., & Pascoal Neto, C. (2004). Isolation andstructural characterization of polysaccharides dissolved in Eucalyptusglobulus kraft black liquors. Carbohydrate Polymers, 60, 77-85.Megiatto, J. D., Houreau, Jr. W., Gardrat, C., Frollini, E., Castellan, A. (2007).Sisal fibers: surface chemical modification using reagent obtained fromrenewable source, characterization of hemicellulose and lignin as modelstudy. Journal of Agriculture and Food Chemistry, 55, 8576-8584.Mwaikambo, L. Y. (2006). Review of the history, properties and application ofplant fibers. African Journal of Science and Technology, 7, 120-133.Neto, P. C., Seca, A., Fradinho, D., Coimbra, M. A., Domingues, F., Evtuguin,D., Silvestre, A., & Cavaleiro, J. A. S. (1996). Chemical composition andstructural features of the macromolecular components of Hibiscus cannabinusgrown in Portugal. Industrial Crops and Products, 5, 189-196.Nobel, P. S. (2003). Environmental Biology of Agaves and Cacti. Cambridge:Cambridge Press.Pinto, P. C., Evtuguin, D. V., & Pascoal Neto, C. (2005). Structure of hardwoodglucuronoxylans: modifications and impact on pulp retention during woodkraft pulping. Carbohydrate Polymers, 60, 489-497.Rowell R. M., Han J. S., & Rowell, J. S. (2000). Characterization and factorsaffecting fiber properties. In: Natural Polymers and Agrofibers Composites(Frollini E, Leão AL, Mattoso LHC eds), IQSC/USP, UNESP and EmbrapaInstrumentação Agropecuária. São Carlos - Brazil, pp. 115-134.237

5. Resultados y discusiónSelvendran, R. R., March, J. F. & Ring, S. G., (1979). Determination of aldosesand uronic acids content of vegetable fiber. Analytical Biochemistry, 96, 282-292.Shimizu, K. (1991). Chemistry of hemicelluloses. In: Hon, D. H. -S., Shiraishi,N. (Eds.), Wood and Cellulosic Chemistry. (pp. 3-57), New York: MarcelDekker.Stewart, D., Azzini, A. Hall, A. T., & Morrison, I. M. (1997). Sisal fibres andtheir constituent non-cellulosic polymers. Industrial Crops & Products, 6, 17-26.Sun, R. C., Tomkinson, J., Ma, P. L., & Liang, S. F. (2000). Comparative studyof hemicelluloses from rice straw by alkali and hydrogen peroxide treatments.Carbohydrate Polymers, 42, 111-122.Sundberg, A., Sundberg, K., Lillandt, C., & Holmbom, B. (1996). Determinationof hemicelluloses and pectins in wood and pulp fibers by acid methanolysisand gas chromatography. Nordic Pulp and Paper Research Journal, 4, 216-226.Tappi Test Methods (1993), Atlanta: TAPPI Press.Teleman, A., Hajunpää, V., Tenkanen, M., Buchert, J., Hausalo, T., Drakenberg,T., & Vuorinen, T. (1995). Characterization of 4-deoxy--L-threo-hex-4-enopyranosyluronic acid attached to xylan in pine kraft pulp and pulpingliquor by 1 H and 13 C NMR spectroscopy. Carbohydrate Research, 272, 55-71.Teleman, A., Lundqvist, J., Tjerneld, F., Stalbrand, H., & Dahlman, O. (2000).Characterization of acetylated 4-O-methylglucuronoxylan isolated from aspenemploying 1 H and 13 C NMR spectroscopy. Carbohydrate Research 329, 807-815.Teleman, A.; Tenkanen, M.; Jacobs, A.; & Dahlman, O. (2002).Characterization of O-acetyl-(4-O-methylglucurono)xylan isolated from birchand beech. Carbohydrate Research, 337, 373-377.Torosantucci, A., Bromuro, C., Chiani, P., De Bernardis, F., Berti, F., Galli, C.,Norelli, F., Bellucci, C., Polonelli, L., Costantino, P., Rappuoli, R., &Cassone, A. (2005) A novel glyco-conjugate vaccine against fungalpathogens. The Journal of Experimental Medicine, 202, 597- 606.Vignon, M. R., & Gey, C. (1997). Isolation, 1 H and 13 C NMR studies of (4-Omethyl-D-glucurono)-D-xylansfrom luffa fruit fibers, jute bast fibers andmucilage of quince tree seeds. Carbohydrate Research, 307, 107-111.238

5. Resultados y discusiónVuorinen, T., Fagerström, P., Buchert, J., Tenkanen, M., & Teleman, A. (1999).Selective hydrolysis of hexenuronic acid groups and its application in ECFand TCF bleaching in kraft pulps. Journal of Pulp and Paper Science, 25(5),155-162.239

5. Resultados y discusiónPublicación IX:Marques G., Gamelas J. A., Ruiz-Dueñas F.J., del Río J.C., Evtuguin D.V.,Martínez A.T. and Gutiérrez A. (2010) Delignification of eucalypt kraft pulpwith manganese-substituted polyoxometalate assisted by fungal versatileperoxidase. Bioresource Technology, 101, 5935-5940.240

5. Resultados y discusiónDelignification of eucalypt kraft pulp with manganese-substitutedpolyoxometalate assisted by fungal versatile peroxidaseGisela Marques a , José A.F. Gamelas b, , Francisco J. Ruiz-Dueñas c , José C. del Rio a , DmitryV. Evtuguin b , Angel T. Martínez c , Ana Gutiérrez aa Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, PO Box 1052, E-41080Seville, Spainb University of Aveiro, CICECO, 3810-193, Aveiro, PortugalcCentro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 MadridAbstractOxidation of the manganese-substituted polyoxometalate [SiW 11 Mn II (H 2 O)O 39 ] 6-(SiW 11 Mn II ) to [SiW 11 Mn III (H 2 O)O 39 ] 5- , one of the most selectivepolyoxometalates for the kraft pulp delignification, by versatile peroxidase (VP)was studied. First, SiW 11 Mn II was demonstrated to be quickly oxidized by VP atroom temperature in the presence of H 2 O 2 (K m = 6.4±0.7 mM and k cat = 47±2 s -1 ).Second, the filtrate from eucalypt pulp delignification containing reducedpolyoxometalate was treated with VP/H 2 O 2 , and 95-100% reoxidation wasattained. In this way, it was possible to reuse the liquor from a first SiW 11 Mn IIIstage for further delignification, in a sequence constituted by twopolyoxometalate stages, and a short intermediate step consisting of the additionof VP/H 2 O 2 to the filtrate for SiW 11 Mn II reoxidation. When the first ClO 2 stageof a conventional bleaching sequence was substituted by the two-stagedelignification with polyoxometalate (assisted by VP) a 50% saving in ClO 2 wasobtained for similar mechanical strength of the final pulp.Keywords: Polyoxometalate, versatile peroxidase, oxidative delignification, pulpbleaching, eucalypt kraft pulp1. IntroductionThe residual lignin remaining after wood pulping is the target of the bleachingprocess 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 physicalproperties of the bleached pulp.In the middle of the 1990 ' s, polyoxometalates (POM) were proposed as anenvironmentally-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 thebleaching/delignification of pulps both as reagents under anaerobic conditions(in this case a second stage is required for POM reoxidation and reuse) or ascatalysts under aerobic conditions (Gamelas et al., 2008; Gaspar et al., 2007;241

5. Resultados y discusiónWeinstock et al., 1997). Applied as catalysts, POM oxidizes the residual ligninin pulp, and the reduced form of POM is reoxidized by molecular oxygen at thesame stage. Therefore, it is possible to reuse the POM solutions in a closed loop.The thermodynamic conditions required for lignin oxidation and reoxidation ofthe POM are related to the corresponding redox potentials as follows: E (Lignin)< E (POM) < E (O 2 ) = 1.22 – 0.059 pH.Several POM types, mostly with the Keggin-type structure (Fig. 1), havebeen considered for kraft pulp delignification, such as [SiW 11 VO 40 ] 5- ,[SiW 10 V 2 O 40 ] 6- , “SiW 10.1 Mo 1.0 V 0.9 O 40 ”, and [SiW 11 Mn(H 2 O)O 39 ] 5- (SiW 11 Mn)(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 = +0.7–0.8 V), although lower than oxygen redox potential, are hardlyreoxidized 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, SiW 11 Mn has been found to be highly selective in pulpdelignification (Gamelas et al., 2005a; Gaspar et al., 2003). The SiW 11 Mn/O 2catalytic system has been compared to the conventional alkaline oxygen processalready used by the pulp industry. In addition to lignin removal, an importantadvantage of the SiW 11 Mn-based process, when applied to eucalypt kraft pulps,is the higher reduction of kappa number than in the alkaline oxygen process, dueto the degradation of hexenuronic acids at the low pH used in these reactions(Gamelas et al., 2005a). However, SiW 11 Mn II is very slowly reoxidized underthese conditions, limiting its practical application.Fig. 1. Structural representation of the Mn-substituted polyoxometalate, -[SiW 11 Mn III (H 2 O)O 39 ] 5- . The dark octahedron represents the Mn III O 5 (H 2 O) group with Mn atthe centre of the octahedron.Enzymatic catalysis is a promising approach to regenerate some of the POMspecies that are difficult to be reoxidized by O 2 and other chemical oxidizers. Inthis context, fungal laccase (from Trametes versicolor) has been assayed for thereoxidation of [SiW 11 V IV O 40 ] 6- and [SiW 11 Mn II (H 2 O)O 39 ] 6- (SiW 11 Mn II )242

5. Resultados y discusión(Gamelas et al., 2005b; Tavares et al., 2004). Although laccase easily oxidizedV IV to V V in the former POM, the corresponding oxidation of Mn II to Mn III inthe manganese-based POM was slow (less that 50% after 4 h at 45 ºC, and under0.3 bar oxygen pressure) (Gamelas et al., 2005b). This urged the search foralternative faster methods of oxidation of Mn II -substituted POM.In contrast to laccase, versatile peroxidase (VP) produced by fungi of thegenera Pleurotus and Bjerkandera is a high redox-potential enzyme able tooxidize a variety of substrates, including free Mn II , due to the presence ofdifferent catalytic sites in its molecular architecture (Ruiz-Dueñas et al., 2009).VP is activated by H 2 O 2 in a two-electron reaction yielding highly reactiveintermediate states. Activated VP can oxidize two substrate molecules in twosuccessive one-electron reactions. It has been demonstrated that Mn III , resultingfrom Mn II oxidation by VP or related manganese peroxidase (Ruiz-Dueñas etal., 2007), is stabilized in solution by the chelation of dicarboxylic acids of smallsize produced by ligninolytic fungi. The manganic cation can, then, act as anoxidizer of lignin contributing to wood delignification in nature (Wariishi et al.,1992).In the present work, reoxidation of the Mn II -containing POM, SiW 11 Mn II , bythe VP/H 2 O 2 system, was studied for the first time. Based on the easy oxidationof Mn II (as a free ion or in POM complexes) by the enzyme a novel approach forthe delignification catalysis was developed. Reduced POM in the liquor from afirst eucalypt pulp delignification stage was reoxidized by VP, and the resultantliquor mixed with the partially delignified pulp for a further delignification stagein a simple POM-VP-POM trial. In addition, delignification of eucalypt pulp byPOM in a VP-assisted process was tested as a pre-bleaching stage to substitutethe first Cl 2 O stage in a conventional elemental chlorine free (ECF) bleachingsequence.2. Materials and methods2.1. Pulp samples and POM synthesisThe delignification assays were carried out with Eucalyptus globulusunbleached kraft pulp supplied by ENCE pulp mill (Spain). The pulp had akappa number of 13.7, and an intrinsic viscosity of 1180 cm 3 /g.For the kinetic studies of SiW 11 Mn II oxidation by VP/H 2 O 2 , the potassiumsalt of the Mn II -containing POM, K 6 [SiW 11 Mn II (H 2 O)O 39 ].10 H 2 O, was prepared(Tourné et al., 1970). For the delignification experiments, a solution containing2.8 0.1 mmol/L of [SiW 11 Mn III (H 2 O)O 39 ] 5- (SiW 11 Mn III ) was prepared aspreviously reported (Galli et al., 2007).2.2. VP expression, in vitro activation and purificationRecombinant VP was obtained from E. coli W3110 transformed with thepFLAG-VPL2 expression vector as previously described (Pérez-Boada et al.,243

5. Resultados y discusión2002). The enzyme was activated in vitro after solubilization of inclusion bodiesin 8 M urea. The folding conditions included 0.15 M urea, 5 mM Ca 2+ , 20 μMhemin, a 4:1 oxidized-glutathione/reduced-glutathione ratio and 0.1 mg/mLprotein at pH 9.5. The active enzyme was purified in a single chromatographicstep (Resource-Q column, Pharmacia-Biotech) using a 0–0.3 M NaCl gradient(2 mL/min) in 10 mM sodium tartrate (pH 5.5) containing 1 mM CaCl 2 . Theconcentration of the enzyme was determined by spectrophotometry ( 407 150000 M -1 cm -1 ) (Ruiz-Dueñas et al., 1999).2.3. SiW 11 Mn II oxidation by VPOxidation of the Mn II -substituted POM was followed at 20 ºC in a quartz cuvette(1 cm optical path) under stirring: 3.0 mL of 0.1 M acetate solution (pH 4.5)containing 2.7 mM SiW 11 Mn II (K 6 [SiW 11 Mn II (H 2 O)O 39 ].10H 2 O), 0.56-1.26 MVP and 0.57-2.24 mM H 2 O 2 were mixed inside the cell. The increase ofabsorbance at 490 nm was followed for 1 min, until a constant value wasreached. The oxidation degree was estimated using the molar extinctioncoefficients of the oxidized and reduced POM (SiW 11 Mn III 490 325 M -1 cm -1 ;and SiW 11 Mn II 490 22 M -1 cm -1 ) (Tourné et al., 1970). For the assays with thedelignification liquor, the H 2 O 2 amount varied between 0 and 2.06 mM, with theamount of enzyme kept at 1.20 M. The total absorbance was corrected for theliquor contribution.Steady-state kinetic constants were calculated during VP oxidation ofincreasing SiW 11 Mn II concentrations in 0.1 M sodium tartrate, pH 5, containing0.1 mM H 2 O 2 . The enzymatic activity at 20 ºC was measured as the initialvelocity, taking linear increments. Mean values and standard errors for theapparent affinity constant (Michaelis constant, K m ) and maximal enzymeturnover (catalytic constant, k cat ) were obtained by non-linear least-squaresfitting of the experimental measurements to the Michaelis-Menten model.Fitting of these constants to the normalized equation v = (k cat /K m )[S]/(1+[S]/K m )yielded the efficiency value (k cat /K m ) with its corresponding standard error.2.4. Pulp delignification experimentsPulp delignification was carried out in a PARR reactor, model 4843 (0.25 L)equipped with an automatic temperature control system, pressure control andmechanical stirring (220 rpm). 7.5g of pulp (dry weight), 67 mL of 0.2 Msodium acetate (pH 4.5), 13 mL of 28 mM POM (SiW 11 Mn III ) solution, andwater to make a final volume of 132 mL were put inside the reactor. The finalconcentration of POM was 2.7 mM. At the end of the reactions, the reactor wasquickly cooled with water and degasified.In the two-stage experiments, including intermediate POM reoxidation withVP (POM-VP-POM reox ), the pulp from the first stage was filtered and pressed,the required amounts of enzyme and H 2 O 2 were added to the delignification244

5. Resultados y discusiónliquor, and the solution was stirred at 20-25 ºC for 10 min. The liquor containingthe reoxidized POM (verified by visible absorption spectroscopy) was mixedagain with the filtered pulp and a second delignification stage was applied underthe same experimental conditions of the first stage. A two-stage experiment notincluding the reoxidation step of POM by VP/H 2 O 2 was also performed byadding fresh POM (SiW 11 Mn III ), acetate buffer and water to the washed pulpobtained after the first stage.After each delignification sequence the pulps were filtered and washed withwater until neutral filtrate. Alkaline extraction of pulps was carried out at 70 ºCduring 1 h and NaOH charge of 2% (on the dry pulp weight).2.5. Modified ECF bleaching sequenceBleaching with Cl 2 O was performed on untreated kraft pulp and with pulpdelignified with POM, at 10% pulp consistency, in plastic bags in a Grant modelY28 thermostatic bath. Two bleaching sequences, D-Ep-D-Ep-D and POM-VP-POM reox E-D-Ep-D, were studied (D refers to Cl 2 O stage; Ep to peroxidereinforcedalkaline extraction, POM-VP-POM reox corresponds to VP-assistedtwo stage [2 h + 2 h] POM treatment; and E to alkaline extraction). Thebleaching conditions in the D-Ep-D-Ep-D sequence were as follows: first Dstage at 50 ºC for 1 h; second D stage at 70 ºC for 2 h; third D stage at 70 ºC for2.5 h; first Ep stage at 70 ºC for 1 h, using 2.0% NaOH and 0.2% H 2 O 2 ; secondEp stage at 70 ºC for 1 h, using 1.5% NaOH and 0.1% H 2 O 2 .The pulp delignified with POM (2 h)-VP-POM reox (2 h) and extracted withNaOH was subjected to D-Ep-D bleaching (POM-VP-POM reox E-D-Ep-Dsequence). The conditions of the last stages in this sequence were as follows:first D stage at 50 ºC for 1 h; second D stage at 70 ºC for 2.5 h; Ep stage at 70 ºCfor 1 h, using 1.5% NaOH and 0.2% H 2 O 2 . The loads of ClO 2 for each stage inboth sequences are discussed in the text.2.6. Pulp characterizationThe treated pulps were characterized using TAPPI T236 cm–99 standard for thekappa number (Tappi, 2006), and the SCAN-CM 15:88 standard for the intrinsicviscosity (Scandinavian Pulp Paper and Board Committee, 1994). Hexenuronicacid content was determined by acid hydrolysis in sodium formate (pH 3.0)followed by spectrophotometric (245 nm) quantitation of the furan derivativesformed (Vuorinen et al., 1999). The acid hydrolysis treatment of pulp wascarried out in the same PARR reactor mentioned above (see 2.4). The strengthproperties, brightness, and opacity of the bleached pulps were determinedaccording to ISO (International Organisation for Standardization Documentationand Information, 2003) and TAPPI (Tappi, 2006) standards.245

5. Resultados y discusión3. Results and discussion3.1. Kinetics of SiW 11 Mn II oxidation by VPThe ability of VP, a peroxidase acting on free Mn II and other substrates, tooxidize this metal ion in Mn-substituted POM is demonstrated here for the firsttime. The steady-state kinetic constants for SiW 11 Mn II oxidation, obtained bynon-linear fitting of initial velocities vs substrate concentration (Fig. 2), revealedhigh VP turnover on SiW 11 Mn II , with a k cat of 47 ± 2 s -1 , and a moderate affinityfor this compound, with a K m of 6.4 ± 0.7 mM. This VP activity was lower thanon free Mn II , with a reported k cat value near 300 s -1 , but the main differencebetween both substrates concerned K m that was around 0.19 mM for free Mn II ,revealing over 30-fold higher affinity of VP on the free metal ion (Ruiz-Dueñaset al., 2007). As a result, the global catalytic efficiency of VP oxidizingSiW 11 Mn II (7.36 ± 0.6 mM -1 s -1 ) was around 200-fold lower than that foroxidation of free Mn II (1600 ± 100 mM -1 s -1 ).4030k obs (s -1 )2010K m (6.4 ± 0.7) mMk cat (47.3 ± 2) s-1Efficiency (7.36 ± 0.6) mM -1 s -100 5 10 15 20 25 30[SiW 11 Mn II ] (mM)Fig. 2. Michaelis-Menten kinetics of SiW 11 Mn II oxidation by VP. The K m , k cat and efficiencyvalues (means and standard errors) are shown inside the plot.The high affinity of VP for free Mn II is due to the existence of a specificcatalytic site in this enzyme constituted by three acidic residues forming a smallchannel on the internal heme propionate, where the free metal cation is oxidized(Ruiz-Dueñas et al., 2007). The lower efficiency observed for SiW 11 Mn IIoxidation by VP was in the order of those reported both for veratryl alcoholoxidation taking place at a tryptophan residue exposed to the solvent (Pérez-Boada et al., 2005), and for oxidation of phenols at the edge of the main hemeaccess channel (Ruiz-Dueñas et al., 2008). This suggested that SiW 11 Mn II couldbe oxidized in one of these two easily accessible catalytic sites, and not in thenarrow channel described for free Mn II that most probably provides a limitedaccess to the bulky SiW 11 Mn II . A detailed kinetic study of different VP variantsmutated at the three catalytic sites mentioned above would be necessary todefinitively identify the SiW 11 Mn II oxidation site in VP.246

5. Resultados y discusión3.2. Optimization of SiW 11 Mn II oxidation by VP (in the presence of H 2 O 2 )A set of assays was carried out aiming to optimize the oxidation of SiW 11 Mn II toSiW 11 Mn III by VP (in the presence of H 2 O 2 ), either by using an aqueous solutionof SiW 11 Mn II buffered at pH 4.5, and the liquor from previous eucalypt kraftpulp delignification with POM (Table 1). The assays were performed at 20 ºC,with 2.7 mM POM concentration, and varying the H 2 O 2 /POM and POM/VPmolar ratios. During the oxidation of POM by VP (and H 2 O 2 ) no indicationabout the formation of other species besides SiW 11 Mn III was obtained.Table 1. Oxidation of SiW 11 Mn II by VP/H 2 O 2 in two distinct reaction media usingdifferent H 2 O 2 , POM and VP molar ratios aH 2 O 2 /POM H 2 O 2 /VP POM/VP Oxidation (%) Time (min)A) Oxidation of SiW 11 Mn II in aqueous solution buffered at pH 4.50.19 446 2382 40 20.40 883 2226 78 20.50 1117 2226 95 50.61 1351 2226 100 50.81 1793 2226 57 220.50 2495 5009 55 290.50 b 2495 5009 94 10B) Oxidation of SiW 11 Mn II in the filtrate from one-stage delignification with POM0 0 2227 18 10.21 472 2227 57 20.40 896 2227 93 60.50 1116 2230 97 20.59 1321 2227 74 270.79 1769 2227 44 18a The assays were performed at 20 ºC, with 2.6-2.8 mM POM concentrationb H 2 O 2 was added in five portions each of them including 20% of the total volume requiredIn the assays performed with the SiW 11 Mn II solution (Table 1A), the extentof POM oxidation (for a fixed amount of enzyme) increased with the H 2 O 2 /POMratio until a 0.5-0.6 molar ratio, and then decreased at higher ratios. Using thisH 2 O 2 /POM ratio (0.5-0.6), 95-100% POM oxidation was obtained in less than 5min, with a POM/VP ratio ~2200. These values were in agreement with thestoichiometry of the global enzymatic reaction, which predicts that 0.5 moles ofH 2 O 2 will be needed to oxidize 1 mole of SiW 11 Mn II . For the H 2 O 2 /POM ratio of0.8, only 57% oxidation of SiW 11 Mn II was obtained, indicating enzymeinactivation by the excess of H 2 O 2 (Valderrama et al., 2002). If the amount ofenzyme was reduced to about 50%, keeping the H 2 O 2 /POM ratio of 0.5, theoxidation extent also decreased (to only 55%) due to the increased H 2 O 2 /VPratio. However, when the later assay was carried out by adding the H 2 O 2 inseveral steps, without exceeding a 500-fold molar excess of H 2 O 2 in eachaddition, the extent of oxidation (94%) was similar to that attained using ahigher amount of enzyme. These data confirmed VP inactivation by H 2 O 2 (even247

5. Resultados y discusiónin the presence of enough amount of SiW 11 Mn II to consume all the H 2 O 2 ) andshowed that the enzyme dose can be reduced by stepwise addition of H 2 O 2 (toprevent VP inactivation). No POM oxidation was observed in the absence ofH 2 O 2 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 varyingthe H 2 O 2 /POM ratio between 0 and 1, the highest POM oxidation degrees (over90%) were obtained at the H 2 O 2 /POM ratios of 0.4-0.5. In the absence of H 2 O 2 ,some POM oxidation occurred suggesting that some substances present in thedelignification liquor may act as the enzyme oxidizing agents. In fact, for all theassays performed with a H 2 O 2 /POM ratio up to 0.5, the oxidation of POM washigher when the reactions were conducted in the delignification liquor thanthose in the SiW 11 Mn II aqueous solutions. Again, the use of H 2 O 2 /POM ratios 0.6 did not improve POM reoxidation in the delignification liquor, and lowerrates were obtained. It was concluded that a H 2 O 2 /POM ratio around 0.5 and aPOM/VP ratio of 2000-3000 should be used to obtain near complete oxidationof the manganese-substituted POM.3.3. Two-stage POM delignification of pulp assisted by VPAs a continuation of the above studies, which showed easy oxidation ofSiW 11 Mn II by VP/H 2 O 2 , a novel approach for the paper pulp delignification wasdeveloped. A first delignification stage using chemically-prepared SiW 11 Mn IIIand O 2 was followed by pulp filtration, and a short intermediate step consistingof the addition of VP and H 2 O 2 to the filtrate. Fig. 3 shows the visible absorptionspectra of the filtrate before and after the enzymatic treatment (resulting in thecomplete reoxidation of the previously reduced POM). Then, the filtrate withreoxidized POM was mixed again with the pulp, and a second delignificationstage (under the same conditions of the first one) was applied. The results werecompared with those obtained when the second delignification stage wasperformed by adding chemically-prepared SiW 11 Mn III , as well as when onlyone-stage POM delignification was performed (Table 2).After one-stage POM delignification at 110 ºC, decreases of kappa number (arough measure of the lignin content in pulp) of 40% and 50%, with viscositylosses of only 3% and 6%, were obtained after 1-h and 2-h reaction, respectively(Table 2). Besides residual lignin, hexenuronic acids contribute significantly tothe kappa number in E. globulus kraft pulps and to the consumption of bleachingreagents (Furtado et al., 2001). In fact, a significant removal of hexenuronicacids (up to 70% after 2 h) was detected after the POM treatment. It isnoteworthy that the POM/O 2 system was highly selective for delignificationwhen compared with the oxygen-delignification control, which showed aviscosity loss of 28% (near 10-fold higher than that obtained with POMdelignification) (Gamelas et al., 2005a).248

5. Resultados y discusión1,5Absorbance10,5ba0300 400 500 600 700 800Wavelength (nm)Fig. 3. UV-vis spectra of the filtrate after 2-h treatment of eucalypt kraft pulp with POM/O 2(a), and after short incubation of this delignification liquor with VP and H 2 O 2 at 20-25ºC (b),revealing the typical SiW 11 Mn II and SiW 11 Mn III spectra, respectively.After two-stage (2-h each) POM delignification including the intermediatereoxidation step with VP and H 2 O 2 , kappa number was reduced of 62% and theviscosity had a drop of 11% (Table 2). Interestingly, this treatment alsodegraded almost 90% of the hexenuronic acids present in the pulp. Thedelignification degree corrected for the hexenuronic acids content was of 51%.Similar results in terms of pulp kappa number, viscosity and hexenuronic aciddegradation were obtained in parallel assays in which freshly-preparedSiW 11 Mn III was added after the first POM stage, revealing that the presence ofthe enzyme did not exert a negative effect on the performance and selectivity ofthe SiW 11 Mn III /O 2 system.Table 2. Delignification of eucalypt kraft pulp with SiW 11 Mn III /O 2 assisted by VP/H 2 O 2(effect of different treatments on pulp kappa number, intrinsic viscosity and hexenuronicacid content (HexA)) aKappa Viscosity Kappa Viscositynumber (cm 3 /g) decrease (%) d loss (%)Initial kraft pulp 13.6 1215 - - 61.2O 2 (without POM, 2 h) 7.3 875 46 (33) 28 15.7POM (1 h) 8.2 1180 40 (33) 3 28.5POM (2 h) 6.8 1140 50 (40) 6 18.4POM (1 h)-VP-(1 h) b 6.5 1130 52 (42) 7 16.8POM (2 h)-VP-(2 h) b 5.2 1080 62 (51) 11 9.3POM (2 h)-POM (2 h) c 5.2 1085 62 (50) 11 8.2a Pulp consistency of 5.3%; 2.7 mM POM; pH 4.5; pO 2 of 0.5 MPa; 110 ºC; and 220 rpmb The pulp after the first stage was filtered, and the POM in the filtrate reoxidized by VP/H 2 O 2 .c The pulp after the first stage was washed, and fresh POM (SiW 11 Mn III ) was addedd Kappa number reduction corrected for HexA (kappa cor = kappa - 0.073 [HexA]) in parenthesesHexA(mmol/kg)249

5. Resultados y discusión3.4. Modified ECF bleaching including a VP-assisted POM stagePulp treatment with the above VP-assisted two-stage (2-h each) POMdelignification followed by an alkaline extraction (POM-VP-POM reox -E) wasinvestigated to substitute the first Cl 2 O stage in a conventional D-Ep-D-Ep-DECF bleaching sequence for eucalypt kraft pulp. Results from the conventionalD-Ep-D-Ep-D bleaching sequence (see Materials and methods) and thesequence including VP-assisted two-stage POM delignification, (POM-VP-POM reox E-D-Ep-D), were compared in terms of Cl 2 O savings for the same finalbrightness (~89% ISO). Pulp bleached by the sequence including VP and POMshowed a Cl 2 O consumption 50% lower than the conventional ECF sequence(Table 3). The Cl 2 O oxidation equivalents (OXE) per kappa number unit in themodified sequence were higher than in the conventional sequence. Moreover,the main strength properties of the unbeaten pulps after the two bleachingsequences were very similar (Table 4). The results obtained suggest that VPassistedcontinuous reutilization of SiW 11 Mn III in a two-reactor system (Gamelaset al., 2007) may be implemented in future industrial ECF sequences, with noapparent deterioration of the pulp strength properties, while significantlyreducing the Cl 2 O consumption, and consequently lowering the environmentalimpact of the bleaching process.Table 3. Cl 2 O consumption and oxidation equivalents (OXE) for eucalypt pulpbleaching in a conventional ECF sequence and after substituting the first D-stage byVP-assisted two-stage POM delignification (89% ISO final brightness)D-Ep-D-Ep-D POM-VP-POM reox E-D-Ep-DClO 2 consumption a 25 + 9 + 6 15 + 5OXE b 90 134a As active chlorine in each D stage (kg/ton)bAs moles of active chlorine per ton of dry pulp and kappa unit4. ConclusionsIn this work, we demonstrate that the reduced Mn-substituted POM, SiW 11 Mn II ,can be oxidized by VP (in the presence of H 2 O 2 ) following Michaelis-Mentenkinetics. This POM, whose oxidized form is highly selective for delignification,was fully oxidized by VP/H 2 O 2 at 20-25 ºC in less than 10 min. In this way, atwo-stage POM delignification process, including a fast intermediate stepconsisting of the addition of VP (and H 2 O 2 ) to the delignification filtrate forPOM reoxidation, was performed, resulting in 62% reduction of the pulp kappanumber and a viscosity loss of only 10%. The substitution of the first Cl 2 O stageby a POM-VP-POM reox treatment in a conventional ECF bleaching sequenceallowed 50% Cl 2 O saving without decreasing the pulp strength properties.250

5. Resultados y discusiónHence, the continuous reutilization of SiW 11 Mn III in a two-reactor system maybe implemented in the future.Table 4. Physical properties of unbeaten bleached pulps (89% ISO, and 65 g/m 2 ) from aconventional ECF sequence and after substituting the first Cl 2 O stage by VP-assisted two-stagePOM delignificationD-Ep-D-Ep-D POM-VP-POM reox -E-D-Ep-DBeating degree (ºSR) 19 20Bulk density (g/cm 3 ) 0.56 0.57Burst index (kPa.m 2 /g) 1.39 1.45Tensile strength (N.m/g) 30.4 28.9Tear index (mN.m 2 /g) 5.2 6.0Elongation (%) 2.2 2.1Stiffness (kN/m) 409 396Opacity (%) 75.8 77.2Internal bonds (Scott test, J/m 2 ) 106 114Air resistance (Gurley-100 mL, s) 0.8 0.8AcknowledgementsThis 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.ReferencesEvtuguin, D.V., Neto, C.P., 1997. New polyoxometalate promoted method ofoxygen delignification. Holzforschung 51, 338-342.Furtado, F.P., Evtuguin, D.V., Gomes, T.M., 2001. Effect of the acid stage inECF 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 ofthe activity of phenoloxidase enzymes. New J. Chem. 31, 1461-1467.Gamelas, J.A.F., Evtuguin, D.V., Gaspar, A.R., 2008. Transition metalcomplexes 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 discusiónGamelas, J.A.F., Gaspar, A.R., Evtuguin, D.V., Neto, C.P., 2005a. Transitionmetal substituted polyoxotungstates for the oxygen delignification of kraftpulp. 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 pulpdelignification. 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 anovel multi-stage process. J. Mol. Catal. B Enzym. 33, 57-64.Gaspar, A., Evtuguin, D.V., Neto, C.P., 2003. Oxygen bleaching of kraft pulpcatalysed by Mn(III)-substituted polyoxometalates. Appl. Cat. A:General239, 157-168.Gaspar, A.R., Gamelas, J.A.F., Evtuguin, D.V., Neto, C.P., 2007. Alternativesfor lignocellulosic pulp delignification using polyoxometalates and oxygen: areview. 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-Mn II 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.Pérez-Boada, M., Doyle, W.A., Ruiz-Dueñas, F.J., Martínez, M.J., Martínez,A.T., Smith, A.T., 2002. Expression of Pleurotus eryngii versatile peroxidasein Escherichia coli and optimisation of in vitro folding. Enzyme Microb.Technol. 30, 518-524.Pérez-Boada, M., Ruiz-Dueñas, F.J., Pogni, R., Basosi, R., Choinowski, T.,Martínez, M.J., Piontek, K., Martínez, A.T., 2005. Versatile peroxidaseoxidation of high redox potential aromatic compounds: Site-directedmutagenesis, spectroscopic and crystallographic investigations of three longrangeelectron transfer pathways. J. Mol. Biol. 354, 385-402.Ruiz-Dueñas, F.J., Martínez, M.J., Martínez, A.T., 1999. Molecularcharacterization of a novel peroxidase isolated from the ligninolytic fungusPleurotus eryngii. Mol. Microbiol. 31, 223-236.Ruiz-Dueñas, F.J., Morales, M., García, E., Miki, Y., Martínez, M.J., Martínez,A.T., 2009. Substrate oxidation sites in versatile peroxidase and otherbasidiomycete peroxidases. J. Exp. Bot. 60, 441-452.252

5. Resultados y discusiónRuiz-Dueñas, F.J., Morales, M., Pérez-Boada, M., Choinowski, T., Martínez,M.J., Piontek, K., Martínez, A.T., 2007. Manganese oxidation site inPleurotus eryngii versatile peroxidase: A site-directed mutagenesis, kineticand crystallographic study. Biochemistry 46, 66-77.Ruiz-Dueñas, F.J., Morales, M., Rencoret, J., Gutiérrez, A., del Río, J.C.,Martínez, M.J., Martínez, A.T., 2008. Improved peroxidases. Patent (Spain)P200801292.Scandinavian Pulp Paper and Board Committee, 1994. SCAN Test Methods.Sweden.Tappi, 2006. 2006-2007 TAPPI Test Methods. TAPPI Press, Norcoss, GA30092, USA.Tavares, A.P.M., Gamelas, J.A.F., Gaspar, A.R., Evtuguin, D.V., Xavier,A.M.R.B., 2004. A novel approach for the oxidative catalysis employingpolyoxometalate-laccase system: application to the oxygen bleaching of kraftpulp. Catal. Commun. 5, 485-489.Tourné, C.M., Tourné, G.F., Malik, S.A., Weakley, T.J.R., 1970.Triheteropolyanions containing copper(II), manganese(II), or manganese(III).J. Inorg. Nucl. Chem. 32, 3875-3890.Valderrama, B., Ayala, M., Vázquez-Duhalt, R., 2002. Suicide inactivation ofperoxidases and the challenge of engineering more robust enzymes. Chem.Biol. 9, 555-565.Vuorinen, T., Fagerstrom, P., Buchert, J., Tenkanen, M., Teleman, A., 1999.Selective hydrolysis of hexenuronic acid groups and its application in ECFand TCF bleaching of kraft pulps. J. Pulp Paper Sci. 25, 155-162.Wariishi, H., Valli, K., Gold, M.H., 1992. Manganese(II) oxidation bymanganese peroxidase from the basidiomycete Phanerochaetechrysosporium. Kinetic mechanism and role of chelators. J. Biol. Chem. 267,23688-23695.Weinstock, I.A., Atalla, R.H., Reiner, R.S., Moen, M.A., Hammel, K.E.,Houtman, C.J., Hill, C.L., Harrup, M.K., 1997. A new environmentallybenign technology for transforming wood pulp into paper. Engineeringpolyoxometalates as catalysts for multiple processes. J. Mol. Catal. A Chem.116, 59-84.Weinstock, I.A., Barbuzzi, E.M.G., Wemple, M.W., Cowan, J.J., Reiner, R.S.,Sonnen, D.M., Heintz, R.A., Bond, J.S., Hill, C.L., 2001. Equilibratingmetal-oxide cluster ensembles for oxidation reactions using oxygen in water.Nature 414, 191-195.253

5. Resultados y discusiónPublicación X:Marques, G., Molina, S., Babot, E.D., Lund, H., del Río, J.C. y Gutiérrez, A.Exploring the potential of a fungal manganese-containing for pitch control andpulp delignification. Bioresource Technology (in preparation).254

5. Resultados y discusiónExploring the potential of a fungal manganese-containing for pitch controland pulp delignificationGisela Marques a , Setefilla Molina a , Esteban D. Babot a , Henrik Lund b , José C. del Río a , AnaGutiérrez aa Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, PO Box 1052,E-41080, Seville, Spainb Novozymes A/S, Krogshoejvej 36, DK-2880 Bagsvaerd, DenmarkAbstractThe potential of the lipoxygenase from Gaeumannomyces graminis to removelipophilic extractives from eucalypt and flax pulps is explored. Pulp treatmentswith the lipoxygenase were performed in the presence and absence of linoleicacid, and with and without a subsequent hydrogen peroxide stage. The highestremoval of lipophilic extractives from eucalypt pulp, including conjugatedsterols (about 40% removal), and free sterols (up to 10% removal), was attainedin the lipoxygenase treatment with linoleic acid followed by the peroxide stage.Different degradation patterns were observed among the lipophilic compoundsin flax pulp with the lipoxygenase treatment, although a high removal (from55% to 70%) of all extractives classes, including alkanes, fatty alcohols, andfree and glycosylated sterols, was attained in most cases. Reactions of thelipoxygenase with model lipid mixtures were carried out to better understand thedegradation patterns observed in pulps. Pulp delignification by the lipoxygenasetreatments was also evaluated.Keywords: Lipoxygenase, Gäumannomyces graminis, Fungal enzymes, Paperpulps, Pitch deposits, Lignin removal.1. IntroductionThe non-polar extractable fraction from wood and nonwood fibers, commonlyreferred to as lipophilic extractives, includes fatty and resin acids, fatty alcohols,alkanes, sterols, sterol esters and triglycerides. These lipophilic compounds arethe precursors of the so-called pitch deposits within the pulp and papermanufacturing processes (Back and Allen 2000). Pitch deposition results in lowquality pulp, and can cause the shutdown of pulp mill operations. Specific issuesrelated to pulps with high extractives content include runnability problems, spotsand holes in the paper, and sheet breaks.In addition to physicochemical methods, biological methods including bothenzymes and microorganisms (Gutiérrez et al. 2001a; 2009), have beeninvestigated to control pitch problems in the pulp and paper industry. Lipases,which hydrolyze triglycerides, are applied with success in softwood (mainlypine) mechanical pulping at mill scale (Fujita et al. 1992) and find widespread255

5. Resultados y discusiónuse nowadays. However, pitch problems in most of the chemical and mechanicalprocesses using other raw materials have not yet been solved. Other compounds,such as free and esterified sterols, resin acids, fatty alcohols and alkanes, areresponsible for pitch problems in these processes (del Río et al. 1999; 2000;Gutiérrez and del Río 2005b). In addition to lipases, the use of sterol esteraseshas also been suggested (Barfoed 2000; Calero-Rueda et al. 2004; Kontkanen etal. 2004) because sterol esters are often at the origin of pitch deposits owing totheir high tackiness and resistance to kraft cooking. However, free sterolsreleased by the action of these esterases are as problematic as sterol esters.On the other hand, the modification of some lipophilic extractives by the useof laccases has been reported (Buchert et al. 2002; Molina et al. 2008; Zhang etal. 2000; 2005). In contrast to lipases and sterol esterases, laccases are oxidativeenzymes whose action is directed toward some aromatic compounds (such asphenols and anilines), although their reactivity with some unsaturated lipids wasdemonstrated. The interest on laccases as industrial biocatalysts has increasedafter discovering the effect of some synthetic compounds (Bourbonnais andPaice 1990; Call 1994) expanding the action of laccases to non-phenolicaromatics and, therefore, increasing their potential in the degradation of ligninand other aromatic compounds (Riva 2006; Rodríguez-Couto and Toca 2006;Widsten and Kandelbauer 2008). Moreover, the use of laccases in the presenceof redox mediators has recently been described for the removal of the lipophilicextractives responsible for pitch deposition from wood and nonwood paperpulps (Gutiérrez et al. 2006a; 2006b; 2007; Molina et al. 2008).In addition to laccases, other oxidative enzymes, such as soybeanlipoxygenases have been tested for the degradation of extractives in softwoodthermo-mechanical pulp (Zhang et al. 2007). Earlier work had also suggestedthe use of lipoxygenases to reduce model wood “pitch” mixtures in pulp andpaper (Borch et al. 2003). Lipoxygenases are non-heme iron-containingdioxygenases which catalyze the oxidation of polyunsaturated fatty acids tohydroperoxides. Despite extensive studies on the properties, genetic informationand physiological functions of this group of enzymes, there is a lack ofutilization of these natural abundant enzymes in industrial processing. Thespecific activity of lipoxygenases to degrade linoleic acid leads to a potentialapplication in papermaking processes to degrade wood extractives that haveadverse effects on pulp and paper properties. In the present work, we study thecapability of the lipoxygenase from the fungus Gaeumannomyces graminis, toremove lipophilic extractives from hardwood (eucalypt) and nonwoody (flax)pulps. This enzyme is a manganese-containing lipoxygenase with severalexceptional 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 60ºC) (Su and Oliw1998) which confer great potential of use under mill process conditions. Sincethe oxidation of polyunsaturated fatty acids by lipoxygenase leads to the256

5. Resultados y discusiónformation 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 presenceof linoleic acid was also investigated.2. Materials and methods2.1. Pulp samplesUnbleached kraft pulp from eucalypt (Eucalyptus globulus) wood, with 44%ISO brightness and 13.5 kappa number was obtained from ENCE (Pontevedra,Spain). Unbleached soda/anthraquinone (AQ) pulp from flax (Linumusitatissimum) with 34% ISO brightness and 11 kappa number was provided byCELESA (Tortosa, Spain).2.2. Model lipidsLinoleic acid, cholesteryl linoleate, nonacosane and octacosanol (from Sigma-Aldrich) and sitosterol (from Calbiochem) were used.2.3. LipoxygenaseThe lipoxygenase (GLOX) used was provided by Novozymes (Bagsvaerd,Denmark) and obtained from the fungus G. graminis. GLOX activity was130000 units per mg, where one unit will cause an absorbance increase at 234nm of 0.001 units per min, at pH 7.0 and 30ºC, when linoleic acid is used assubstrate in a reaction volume of 1.0 ml (light path 1 cm).2.4. Enzymatic treatments of paper pulpsPulp treatments with GLOX (10 mg/g eucalypt pulp and 20 mg/g flax pulp)were carried out using 5 g pulp (dry weight) at 1% consistency (w:w) in 100mM monosodium phosphate (pH 7), 30ºC, with oxygen bubbling, in athermostatic shaker at 170 rev/min for 4 h. Pulp treatments were performed inthe presence and absence of linoleic acid (0.1 mg/g pulp). In a subsequent step,pulps at 5% consistency (w:w) were submitted to a bleaching stage using 3%(w:w) H 2 O 2 and 1.5% (w:w) NaOH, both referred to pulp dry weight, at 90ºCfor 2 h. Controls without GLOX were performed. Treated pulp samples wereSoxhlet extracted with acetone (8 h) and the extracts were evaporated to drynessand redissolved in chloroform for gas chromatography/mass spectrometry (GC-MS) and GC analyses. When required, bis(trimethylsilyl)trifluoroacetamide(BSTFA, from Supelco) in the presence of pyridine was used to preparetrimethylsilyl derivatives.257

5. Resultados y discusión2.5. Enzymatic reactions with model lipidsEnzymatic reactions with mixtures of model compounds listed in section 2.2 (1mg) were performed using GLOX (0.1 mg/mg lipid), and Tween 20 asdispersant (1% v/v) in 100 mM monosodium phosphate (pH 7), 30ºC, withoxygen bubbling, in a thermostatic shaker at 100 rev/min for 2h. In a subsequentstep, the model mixtures after the enzymatic reaction were submitted to ahydrogen peroxide stage, adding 50 μl H 2 O 2 at 30% (w:w) and 37.5 μl 5 NNaOH to each reaction flask, at 90ºC and 100 rev/min for 2 h. Controls withoutGLOX were performed.After the enzymatic treatments, the lipid dispersions were immediatelyevaporated, and the reaction products recovered with chloroform: methanol(1:1), dried and redissolved in chloroform for GC and GC-MS analyses. Whenrequired, BSTFA in the presence of pyridine was used to prepare trimethylsilylderivatives.2.6. GC and GC-MS analyses of lipidsThe GC analyses of lipids from both the enzymatic treatments of pulps andenzymatic reactions with model compounds were performed in an Agilent6890N Network GC system using a short fused-silica DB-5HT capillary column(5 m x 0.25 mm internal diameter, 0.1 m film thickness) from J&W Scientific,enabling simultaneous elution of the different lipid classes (Gutiérrez et al.1998). The temperature program was started at 100°C with 1 min hold, and thenraised to 350°C at 15°C/min, and held for 3 min. The injector and flameionizationdetector (FID) temperatures were set at 300°C and 350°C,respectively. Helium (5 ml/min) was used as carrier gas, and the injection wasperformed in splitless mode. Peaks were quantified by area, and data fromreplicates were averaged. In all cases the standard deviations were below 7% ofthe mean values.The GC-MS analyses were performed with a Varian 3800 chromatographcoupled to an ion-trap detector (Varian 4000) using a medium-length (12 m)capillary column of the same characteristics described above for GC/FID. Theoven was heated from 120°C (1 min) to 380°C at 10°C/min, and held for 5 min.In all GC/MS analyses, the transfer line was kept at 300°C, the injector wasprogrammed from 120°C (0.1 min) to 380°C at 200°C/min and held until theend of the analysis, and helium was used as carrier gas at a rate of 2 ml/min.Compounds were identified by mass fragmentography, and by comparing theirmass spectra with those of the Wiley and NIST libraries and standards.2.7. Pulp evaluationPulp brightness, kappa number and intrinsic viscosity were analyzed followingISO 3688:1999, ISO 302:1981 and ISO 5351/1:1981 standard methods,258

5. Resultados y discusiónrespectively (International Organisation for Standardization Documentation andInformation (ISO) 2003).3. Results and discussion3.1. Composition of lipophilic extractives in eucalypt and flax pulpsThe lipophilic extractives from eucalypt and flax pulps were analyzed by GCand GC-MS (Fig. 1). The compounds from unbleached eucalypt kraft pulp(Fig.1a) include sterols (predominantly sitosterol) in free (peak 8) andconjugated form, both as glycosides (peak 10) and esters (peak 11), as well asfatty acids, mainly palmitic acid (peak 1). The detailed composition of lipophilicextractives from eucalypt pulp has been published elsewhere (Gutiérrez et al.2001b; Gutiérrez and del Río 2001). Among these compounds, free andesterified sterols have been reported to be the main responsible for pitchdeposition during the manufacturing of eucalypt pulp (del Río et al. 1998; 1999;2000). On the other hand, fatty alcohols including hexacosanol (peak 6),octacosanol (peak 7), and triacontanol (peak 9), and free sterols with sitosterolpredominating (peak 8) and fatty acids, predominantly palmitic acid (peak 1),linoleic acid (peak 2), oleic acid (peak 3) and stearic acid (peak 4) are the mainlipophilic extractives identified in flax pulp (Fig.1b). Minor amounts of alkanessuch as nonacosane (peak 5) and sterol glycosides (peak 10) were also present.The detailed characterization of lipophilic extractives from flax pulp has alreadybeen reported (Gutiérrez and del Río 2003a; 2003b; Marques et al. 2010). Fattyalcohols and alkanes have been shown as the main responsible for pitchproblems during manufacturing of nonwoody soda pulps (Gutiérrez and del Río2005a).From the analysis of pulp extractives it would not be expected to see asignificant change in their total content after treatment with GLOX given thatthe amount of extractives with the 1-cis, 4-cis-pentadiene (i.e. linoleic acid) isquite scarce. Still it was chosen to pursue testing of GLOX on both pulps andmodel substrates since other studies (Gutiérrez et al. 1999) had shown thepresence of linoleic acid esterified with sitosterol as the main sterol ester amongeucalypt extractives and, at the same time, other studies had surprisingly shownan ability of lipoxygenase to significantly impact other components inunbleached eucalypt kraft pulp (Borch et al. 2003).3.2. Removal of pulp lipophilic extractives by lipoxygenase treatmentThe eucalypt and flax pulps were treated with the lipoxygenase from G.graminis (GLOX) to evaluate the potential of this enzyme to remove lipophilicextractives from these pulps. Additional assays adding linoleic acid to theenzymatic reactions were also performed to study the mediating effect ofperoxidation products on the lipid removal. After the enzymatic treatments, thepulps were subsequently submitted to a hydrogen peroxide bleaching stage,259

5. Resultados y discusiónwhich is a common stage in the bleaching of these alkaline pulps. Thecomposition of the lipophilic extractives in pulps after the treatment with GLOXwas analyzed by GC and GC-MS and compared with that of control pulp treatedunder the same conditions but without enzyme addition.100%8Relative response12+341011(a)2 4 6 8 10 12 14 16 18 20Retention time (min)100%7Relative response12+345689(b)2 4 6 8 10 12 14 16 18 20Retention 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, sterolglycosides; and 11, sterol esters103.2.1. Eucalypt pulp treatmentsTable 1 shows the percentage of degradation of the main eucalypt pulpextractives after the enzymatic treatment with GLOX. GLOX treatmentproduced a removal of 22% and 20% of the sterol esters and sterol glycosides,respectively, but the amount of free sterols remained unchanged. A similar lackof degradation of free sterols was also observed after TMP pulp treatment withsoybean lipoxygenase (Zhang et al. 2007).260

5. Resultados y discusiónTable 1. Removal (percentage of reduction) of the main lipophilic extractives fromeucalypt pulp after treatment with lipoxygenase (GLOX) in the absence and presenceof linoleic acid (LA), without and with a subsequent H 2 O 2 stage (P).Without H 2 O 2 With H 2 O 2GLOX GLOX/LA GLOX-P GLOX/LA-PFree sterols 0 0 0 7Sterol glycosides 22 24 20 39Sterol esters 20 29 27 40Accordingly it appears that the effect of the lipoxygenase treatment canextend well beyond the primary substrates of the enzyme via the fatty acidhydroperoxides. When the GLOX treatments were performed in the presence oflinoleic acid, a higher decrease of sterol esters and glycosides (up to 24% and29%, respectively) was observed but the content of free sterols was notmodified. On the other hand, in the GLOX treatments followed by a hydrogenperoxide stage, the addition of linoleic acid to the enzymatic reaction caused ahigher removal of sterol esters (from 20% to 39%) and sterol glycosides (from27% to 40%). A slight decrease of free sterols (from 0 to 7%) was alsoobserved.To get further insight into these enzymatic reactions, GLOX reactions withmodel compounds (Fig. 2) representative for the main lipophilic extractives ineucalypt pulp, including free and esterified sterols (sitosterol and cholesteryllinoleate, respectively), and free fatty acids (linoleic acid) were carried out. Thereactivity of the different lipids was studied by GC and GC-MS. The content ofcholesteryl linoleate decreased about 20% while the content of free sitosterolremained unchanged by the GLOX treatment. In addition, linoleic acid wascompletely degraded, as expected. When the GLOX reactions were performedwith a mixture of the three model compounds, reductions of 83% and 25% ofcholesteryl linoleate and sitosterol, respectively, were observed in the reactionsof lipoxygenase, suggesting that peroxidation reactions could mediate the cooxidationof these model lipids. The co-oxidation of sitosterol observed in thereaction of GLOX with the model mixture was not observed in the eucalypt pulptreatment. One potential reason for the limited effect of the treatment on the freesterols may be the predominant localization of these compounds inside the pulpelements (Speranza et al. 2002).In the reaction of lipoxygenase with mixtures of the three model lipids,oxidative derivatives of cholesteryl linoleate and free sitosterol were observed,and these were more evident after a hydrogen peroxide stage (Fig. 3). Thechemical structures of the oxidation products identified are shown in Fig. 4. Thereaction products of cholesteryl linoleate were, as mentioned above, cholesta-3,5-dien-7-one (peak 2) and the cholesteryl ester core aldehyde (peak 6).Oxidized derivatives of free sitosterol, namely 7-hydroxysitosterol (peak 4)261

5. Resultados y discusión(and traces of 7-hydroxysitosterol) and 7-ketositosterol (peak 5) can also beobserved in Fig. 3. These oxidized derivatives were also identified in theenzymatic reactions of these model lipids with laccase in the presence of 1-hydroxybenzotriazole (HBT) as redox mediator (Molina et al. 2008).OaOHHObOcOdFigure 2. Chemical structures of the model compounds representative for main paper pulplipophilic extractives used in the enzymatic reactions: (a) linoleic acid; (b) sitosterol; (c)cholesteryl linoleate; (d) nonacosane and (e) octacosanol.eOH3.2.2. Flax pulp treatmentsThe percentage of removal of lipophilic extractives from flax pulp by the GLOXtreatment is shown in Table 2. All lipophilic extractives from flax pulpdecreased significantly in the several GLOX treatments. Surprisingly, thecontent of free sterols in flax pulp decreased up to 55% after the GLOXtreatments, contrasting with the lack of removal in eucalypt pulp where no effecton free sterols was observed. A high removal of alkanes (up to 48%), fattyalcohols (up 60%) and sterol glycosides (up to 65%) was also observed. Thesterols present in flax is likely distributed throughout the surface layers of thefibers alongside the other lipophilic components and may be partially cosolubilizedwith them, following the reaction with the lipoxygenase on the ciscis-pentadienestructures present in flax pulp.The addition of linoleic acid to these enzymatic reactions caused differenteffects upon the different lipophilic compounds. A higher removal of alkanesand sterol glycosides was observed by the addition of linoleic acid whereas thecontrary happened with fatty alcohols and free sterols. In the GLOX treatmentsof flax pulp followed by a hydrogen peroxide stage, the addition of linoleiccaused an increase in the removal of all the lipophilic extractives.262

5. Resultados y discusión100%1 3(a)Relative response74 6 8 10 12 14Retention time (minutes)16100%3(b)Relative response24 5 674 6 8 10 12 14 16Retention time (minutes)Figure 3. Behaviour of a mixture of different model compounds (linoleic acid, sitosterol,cholesteryl linoleate) representative for eucalypt pulp lipophilic extractives after enzymatictreatment with lipoxygenase. Shown are the GC chromatograms of silylated modelcompounds before treatment (a), and products after reaction with lipoxygenase (b). Peakidentification: 1, linoleic acid; 2, cholesta-3,5-dien-7-one; 3, sitosterol; 4, 7hydroxysitosterol;5, 7-ketositosterol; 6, cholesteryl 9-oxononanoate; and 7, cholesteryllinoleate.Reactions of GLOX with mixtures of four model compounds (Fig. 2)representative for the main lipophilic extractives in flax pulp, including alkanes(nonacosane), fatty alcohols (octacosanol), free sterols (sitosterol), and free fattyacids (linoleic acid) were also carried out. The GC and GC-MS analyses showedreductions of 100, 51, 65 and 55% of linoleic acid, nonacosane, octacosanol andsitosterol, respectively (Fig. 5) after the GLOX treatment in agreement with theresults observed in pulps, suggesting that peroxidation reactions could mediatethe co-oxidation of these lipids. Similar findings were reported in the removal ofalkanes and fatty alcohols from flax pulps with laccase in the presence of HBTas mediator (Molina et al. 2008). The fact that lipid radicals generated fromperoxidation of unsaturated lipids such as linoleic acid participate in the263

5. Resultados y discusiónoxidation of the less reactive lipophilic compounds can be used as a way toremove lipophilic extractives from pulps where linoleic acid is present usingGLOX.Sitosterol oxidation products:HOfOHOgOHCholesteryl linoleate oxidation products:OOhOHOiFigure 4. Chemical structures of the main oxidized derivatives identified after thelipoxygenase reactions with free sterols (sitosterol) and sterol esters (cholesteryl linoleate): (f)7-ketositosterol; (g) 7-hydroxysitosterol; (h) cholesta-3,5-dien-7-one and (i) cholesteryl 9-oxononanoate.Table 2. Removal (percentage of reduction) of main lipophilic extractives in flax pulpafter treatment with the G. graminis lipoxygenase (GLOX) in the absence and presence oflinoleic acid (LA) without and with a subsequent H 2 O 2 stage (P)Without H 2 O 2 With H 2 O 2GLOX GLOX/LA GLOX-P GLOX/LA-PAlkane (C27) 26 39 21 46Alkane (C29) 48 54 35 55Fatty alcohol (C26) 48 44 45 50Fatty alcohol (C28) 55 44 42 49Fatty alcohol (C30) 61 43 52 52Free sterols 55 30 16 34Sterol glycosides 65 51 45 71264

5. Resultados y discusión3.4. Pulp properties after lipoxygenase treatmentIn addition to the enzymatic removal of lipophilic extractives from pulps, theeffect of the lipoxygenase treatment on some selected properties of the pulpswere assessed, including kappa number (a rough estimation of the lignin contentin pulp), brightness and intrinsic viscosity (an estimation of cellulose integrity).Table 3 shows the results for eucalypt pulp. Pulps with lower kappa number (1.2points decrease) and increased brightness (2.6 and 3.4 points increase, in theabsence and presence of linoleic acid, respectively) were obtained aftertreatment with GLOX and subsequent peroxide stage. No improvement of pulpbrightness (and only a decrease of 0.7-0.8 points in kappa number) was observedwhen the enzymatic treatment was not followed by a hydrogen peroxide stage,revealing the need of a peroxide stage after the lipoxygenase treatment toimprove pulp properties. Pulp viscosity was maintained after the enzymatictreatment although decreased after the peroxide stage.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 H 2 O 2 stage (P), andcontrol without enzymeControl GLOX GLOX/LAinitial P initial P initial PKappa number 13.5 10.7 12.8 9.5 12.9 9.5Brightness (% ISO) 44.0 55.9 43.8 58.5 43.9 59.3Intrinsic viscosity (mL/g) 1140 925 1148 800 1143 767In the case of flax pulp (Table 4), an increase of 0.8 and 1.2 points of ISObrightness was achieved after the enzymatic treatment followed by the hydrogenperoxide stage, in the absence and presence of linoleic acid, respectively (noincrease of brightness was observed before the hydrogen peroxide stage). Nosignificant decrease of kappa number (lower than 1 point) was observed after theenzymatic treatment of flax pulp (before the peroxide stage). After peroxide, thekappa number did not decrease and the viscosity increased.Table 4. Properties of flax pulp treated with the G. graminis lipoxygenase (GLOX) inthe absence and presence of linoleic acid (LA) before and after a H 2 O 2 stage (P), andcontrol without enzymeControl GLOX GLOX/LAinitial P initial P initial PKappa number 9.3 5.1 9.1 5.2 8.6 5.5Brightness (% ISO) 35.1 61.2 35.1 62 35.3 62.4Intrinsic viscosity (mL/g) 787 535 762 648 779 602265

5. Resultados y discusiónThe better delignification values observed in the GLOX treatment of eucalyptpulp compared with those of flax pulp treatment can be related with the differentcomposition and structure of lignin in these two types of pulp. The lignin fromeucalypt pulp is characterized by a high abundance of syringyl units (Ibarra et al.2005; 2007) and, therefore, it is easier to delignify than lignin from flax pulpthat is mainly constituted by guaiacyl units (Camarero et al. 2004).100%124(a)Relative response34 6 8 10 12 14Retention time (minutes)100%(b)Relative response2344 6 8 10 12 14Retention time (minutes)Figure 5. Behaviour of a mixture of different model compounds (linoleic acid, nonacosane,octacosanol, sitosterol) representative for flax pulp lipophilic extractives after enzymatictreatment with lipoxygenase. Shown are the GC chromatograms of silylated modelcompounds before treatment (a), and after reaction with lipoxygenase (b). Peak identification:1, linoleic acid: 2, nonacosane; 3, octacosanol; and 4, sitosterol.4. ConclusionsThe potential of the lipoxygenase from G. graminis to remove lipophilicextractives from a hardwood (eucalypt) and a nonwood (flax) pulp has beenstudied. A removal up to 40% of esterified and glycosylated sterols was266

5. Resultados y discusiónachieved by the GLOX treatment of eucalypt pulps, while only a decrease of10% of free sterols was observed. Higher decreases (up to 70%) of lipophilicextractives from flax pulp were produced by the lipoxygenase treatment,including free sterols that decreased 55%. In addition, some pulp propertieswere determined on the enzymatically treated pulps observing a significantincrease of brightness and decrease of kappa number for eucalypt pulp followingsubsequent peroxide treatment, while only limited brightness enhancement wasobserved for flax pulp. Given the significant alkaline activity of lipoxygenases itis suggested that these may have potential use in brown stock treatment as ameans for reducing down-stream deposition problems as well as reducing theamount of chemicals in the subsequent bleaching stages. Further studies areneeded to gain insight into the chemistry of the reactions of the lipoxygenasewith different lipids, and in the potential applicability of this treatment in thepulp and paper industry.AcknowledgementsThis study has been supported by the Spanish projects BIO2007-28719-E andAGL2008-00709 and the EU project BIORENEW (NMP2-CT-2006-026456).Novozymes (Bagsvaerd, Denmark) is acknowledged for GLOX supply andENCE (Pontevedra, Spain) and CELESA (Tortosa, Spain) for eucalypt and flaxpaper pulp samples, respectively. J. Romero (ENCE) and T. Vidal (UPC,Barcelona, Spain) are acknowledged for pulp properties determination.ReferencesBack EL, Allen LH (2000) Pitch control, wood resin and deresination. TAPPIPress, AtlantaBarfoed M (2000) Methods of hydrolyzing cholesterol esters by using aPseudomonas fragi cholesterol esterase. Patent (International) WO9423052:Borch K, Franks N, Lund H, Xu H, Luo J (2003) Oxidizing enzymes in themanufacturing of paper materials. Patent (USA)US 2003-0124710 A1Bourbonnais R, Paice MG (1990) Oxidation of non-phenolic substrates. Anexpanded role for laccase in lignin biodegradation. FEBS Lett 267:99-102Buchert J, Mustranta A, Tamminen T, Spetz P, Holmbom B (2002)Modification of spruce lignans with Trametes hirsuta laccase. Holzforschung56:579-584Calero-Rueda O, Gutiérrez A, del Río JC, Prieto A, Plou FJ, Ballesteros A,Martínez AT, Martínez MJ (2004) Hydrolysis of sterol esters by an esterasefrom Ophiostoma piceae: Application for pitch control in pulping ofEucalyptus globulus wood. Intern J Biotechnol 6:367-375267

5. Resultados y discusiónCall H-P (1994) Verfahren zur Veränderung, Abbau oder Bleichen von Lignin,ligninhaltigen Materialien oder ähnlichen Stoffen. Patent (International) WO94/29510:Camarero S, García O, Vidal T, Colom J, del Río JC, Gutiérrez A, Gras JM,Monje R, Martínez MJ, Martínez AT (2004) Efficient bleaching of non-woodhigh-quality paper pulp using laccase-mediator system. Enzyme MicrobTechnol 35:113-120del Río JC, Gutiérrez A, González-Vila FJ (1999) Analysis of impuritiesoccurring in a totally-chlorine free-bleached Kraft pulp. J Chromatogr 830:227-232del Río JC, Gutiérrez A, González-Vila FJ, Martín F, Romero J (1998)Characterization of organic deposits produced in the Kraft pulping ofEucalyptus globulus wood. J Chromatogr 823:457-465del Río JC, Romero J, Gutiérrez A (2000) Analysis of pitch deposits produced inKraft pulp mills using a totally chlorine free bleaching sequence. J ChromatogrA 874:235-245Fujita 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):117-122Gutiérrez A, del Río JC (2001) Gas chromatography-mass spectrometrydemonstration of steryl glycosides in eucalypt wood, kraft pulp and processliquids. Rapid Commun Mass Spectrom 15:2515-2520Gutiérrez A, del Río JC (2003a) Lipids from flax fibers and their fate in alkalinepulping. J Agric Food Chem 51:4965-4971Gutiérrez A, del Río JC (2003b) Lipids from flax fibers and their fate in alkalinepulping (Vol 51, pg 4965, 2003). J Agric Food Chem 51:6911-6914Gutiérrez A, del Río JC (2005a) Chemical characterization of pitch depositsproduced in the manufacturing of high-quality paper pulps from hemp fibers.Bioresource Technol 96:1445-1450Gutiérrez A, del Río JC (2005b) Pitch episodes produced during themanufacturing of high-quality paper pulps from hemp fibers. BioresourTechnol (submitted):Gutiérrez A, del Río JC, González-Vila FJ, Martín F (1998) Analysis oflipophilic extractives from wood and pitch deposits by solid-phase extractionand gas chromatography. J Chromatogr 823:449-455Gutiérrez A, del Río JC, González-Vila FJ, Martín F (1999) Chemicalcomposition of lipophilic extractives from Eucalyptus globulus Labill. wood.Holzforschung 53:481-486268

5. Resultados y discusiónGutiérrez A, del Río JC, Ibarra D, Rencoret J, Romero J, Speranza M, CamareroS, Martínez MJ, Martínez AT (2006a) Enzymatic removal of free andconjugated sterols forming pitch deposits in environmentally sound bleachingof eucalypt paper pulp. Environ Sci Technol 40:3416-3422Gutiérrez A, del Río JC, Martínez AT (2009) Microbial and enzymatic controlof pitch in the pulp and paper industry. Appl Microbiol Biotechnol 82:1005-1018Gutiérrez A, del Río JC, Martínez MJ, Martínez AT (2001a) Thebiotechnological control of pitch in paper pulp manufacturing. TrendsBiotechnol 19:340-348Gutiérrez A, del Río JC, Rencoret J, Ibarra D, Martínez AT (2006b) Mainlipophilic extractives in different paper pulp types can be removed using thelaccase-mediator system. Appl Microbiol Biotechnol 72:845-851Gutiérrez A, Rencoret J, Ibarra D, Molina S, Camarero S, Romero J, del Río JC,Martínez AT (2007) Removal of lipophilic extractives from paper pulp bylaccase and lignin-derived phenols as natural mediators. Environ Sci Technol41:4124-4129Gutiérrez A, Romero J, del Río JC (2001b) Lipophilic extractives fromEucalyptus globulus pulp during kraft cooking followed by TCF and ECFbleaching. Holzforschung 55:260-264Hamberg M, Su C, Oliw E (1998) Manganese lipoxygenase - Discovery of abis-allylic hydroperoxide as product and intermediate in a lipoxygenasereaction. J Biol Chem 273:13080-13088Ibarra D, Chávez MI, Rencoret J, del Río JC, Gutiérrez A, Romero J, CamareroS, Martínez MJ, Jiménez-Barbero J, Martínez AT (2007) Lignin modificationduring Eucalyptus globulus kraft pulping followed by totally chlorine freebleaching: A two-dimensional nuclear magnetic resonance, Fourier transforminfrared, and pyrolysis-gas chromatography/mass spectrometry study. J AgricFood Chem 55:3477-3499Ibarra D, del Río JC, Gutiérrez A, Rodríguez IM, Romero J, Martínez MJ,Martínez AT (2005) Chemical characterization of residual lignins fromeucalypt paper pulps. J Anal Appl Pyrolysis 74:116-122International Organisation for Standardization Documentation and Information(ISO) (2003) ISO Standards Collection on CD-ROM. Paper, board and pulps,second edn. ISO, GenevaKapich AN, Korneichik TV, Hatakka A, Hammel KE (2010) Oxidizability ofunsaturated fatty acids and of a non-phenolic lignin structure in the manganeseperoxidase-dependent lipid peroxidation system. Enzyme Microb Technol46:136-140269

5. Resultados y discusiónKontkanen H, Tenkanen M, Fagerstrom R, Reinikainen T (2004)Characterisation of steryl esterase activities in commercial lipase preparations.J Biotechnol 108:51-59Marques G, del Rio JC, Gutiérrez A (2010) Lipophilic extractives from severalnonwoody lignocellulosic crops (flax, hemp, sisal, abaca) and their fate duringalkaline pulping and TCF/ECF bleaching. Bioresource Technol 101:260-267Molina S, Rencoret J, del Río JC, Lomascolo A, Record E, Martínez AT,Gutiérrez A (2008) Oxidative degradation of model lipids representative formain paper pulp lipophilic extractives by the laccase-mediator system. ApplMicrobiol Biotechnol 80:211-222Prigge ST, Boyington JC, Faig M, Doctor KS, Gaffney BJ, Amzel LM (1997)Structure and mechanism of lipoxygenases. Biochemie 79:629-636Riva S (2006) Laccases: blue enzymes for green chemistry. Trends Biotechnol24:219-226Rodríguez-Couto S, Toca JL (2006) Industrial and biotechnological applicationsof laccases: A review. Biotechnol Adv 24:500-513Speranza M, Martínez MJ, Gutiérrez A, del Río JC, Martínez AT (2002) Woodand pulp localization of sterols involved in pitch deposition using filipinfluorescent staining. J Pulp Paper Sci 28:292-297Su C, Oliw EH (1998) Manganese lipoxygenase - Purification andcharacterization. J Biol Chem 273:13072-13079Widsten P, Kandelbauer A (2008) Laccase applications in the forest productsindustry: A review. Enzyme Microb Technol 42:293-307Zhang X, Nguyen D, Paice MG, Tsang A, Renaud S (2007) Degradation ofwood extractives in thermo-mechanical pulp by soybean lipoxygenase.Enzyme Microb Technol 40:866-873Zhang X, Renaud S, Paice M (2005) The potential of laccase to removeextractives present in pulp and white water from TMP newsprint mills. J PulpPaper Sci 31:175-180Zhang X, Stebbing DW, Saddler JN, Beatson RP, Kruus K (2000) Enzymetreatments of the dissolved and colloidal substances present in mill white waterand the effects on the resulting paper properties. J Wood Chem Technol20:321-335270

5. Resultados y discusión271

6. ConclusionesCONCLUSIONESEn la presente Tesis se ha estudiado la composición química de losprincipales constituyentes (lignina, lípidos y hemicelulosas) de diferentescultivos lignocelulósicos utilizados como materia prima para la fabricación depastas de celulosa de alta calidad, así como su comportamiento durante losprocesos de pasteado y blanqueo. Además, se han estudiado dos procedimientosbiotecnológicos que incluyen el uso de enzimas fúngicas para la eliminación delignina y lípidos residuales en pastas. Las principales conclusiones obtenidas secitan a continuación:1. En general, las materias primas estudiadas se caracterizan por presentarun alto contenido en polisacáridos y bajo en lípidos y lignina, lo que es,en principio, favorable para el proceso de producción de pasta decelulosa.2. El contenido y composición de las diferentes clases de lípidos varíaentre las distintas fibras estudiadas. Las fibras provenientes de tallos secaracterizan por un alto contenido en ácidos grasos y, en particular, lasfibras de lino, kenaf y yute poseen también un alto contenido en ceras.Por otro lado, las fibras provenientes de hojas además de tener un altocontenido en ácidos grasos, poseen un alto contenido en esteroles (sisaly abacá) y ceras (curauá).3. Las diferentes clases de lípidos muestran distinto comportamientodurante los procesos de cocción y blanqueo. Durante la cocción sosa-AQ, los compuestos esterificados se hidrolizan, los ácidos grasos sedisuelven y los aldehídos se eliminan, mientras que los compuestosneutros (esteroles, alcanos, alcoholes, cetonas) sobreviven a la cocción.Los esteroles libres que sobreviven a la cocción alcalina se degradan enel blanqueo ECF mientras que permanecen prácticamente inalterados enel blanqueo TCF, pudiendo originar problemas de deposición de pitch enestos procesos. Los glicósidos de esteroles, por otra parte, se eliminantanto en el blanqueo TCF como ECF. Los ácidos grasos saturados,alcoholes y alcanos sobreviven tanto a la cocción como al blanqueo ECFy TCF, y por tanto pueden originar también problemas de pitch.4. La composición de las ligninas de las fibras liberianas de kenaf y yute,así como las de todas las fibras de hojas (sisal, abacá y curauá) sonfundamentalmente de tipo S. Por el contrario, la lignina de las fibras decáñamo, lino y caña común tienen un predominio de unidades de tipo G.A pesar de que las fibras de lino y cáñamo presentan un bajo contenido273

6. Conclusionesen lignina (< 5%), son más difíciles de deslignificar puesto la lignina detipo G es más condensada.5. Los principales enlaces entre las unidades de lignina en todas las fibrasestudiadas son de tipo aril-éter -O-4. También están presentes unidadesde tipo resinol -, fenilcumarano -5/-O-4 y espirodienona -1/-O-. La mayor proporción de enlaces -O-4 se encuentra en las ligninasdel kenaf, sisal, abacá y curauá, las cuales al tener también mayorproporción de unidades S, son más fácilmente deslignificables.6. Las ligninas de kenaf, sisal, abacá y curauá están extensamente aciladasen el carbono de la cadena lateral (con grupos acetatos y/o p-cumaratos) y principalmente sobre unidades S. Se demostró que laacilación de la lignina tiene lugar a nivel de monómero y que el sinapilacetato (y otros monómeros acilados) se comportan como auténticosmonómeros de la lignina. Además, estas ligninas están muy enriquecidasen unidades S y en enlaces -O-4, y con poca presencia de enlacescondensados, lo que las hace extremadamente lineales.7. Las hemicelulosas de las fibras liberianas presentan una mayorvariabilidad en cuanto a su composición en azúcares neutros que lasfibras procedentes de hojas. Así, mientras que en las fibras de lino ycáñamo predominan la manosa y la galactosa, en las de kenaf y yute elmonosacárido predominante es la xilosa. Por otro lado, en todas lasfibras de hojas estudiadas (sisal, abacá y curauá) se observó unpredominio de xilosa.8. Las hemicelulosas de sisal se caracterizan por estar constituidasfundamentalmente por un glucuronoxilano acetilado, cuya cadenaprincipal está formada por unidades de -D-xilopiranosa parcialmenteramificada con residuos glucuronosilos (MeGlcpA y GlcpA). El 61%mol. de las unidades de xilopiranosa están acetiladas, principalmente enla posición O-3 y O-2 (39% mol. y 13% mol. respectivamente) y diacetiladas(9% mol.) también en esas posiciones.9. El heteroxilano de sisal sufre una despolimerización y unadesacetilación significativas durante el proceso de pasteado, en el quelos residuos MeGlcpA y GlcpA se eliminan parcialmente o seconvierten en HexA y los grupos acetilo se hidrolizan mayoritariamente.Durante el proceso de blanqueo, una pequeña fracción de los xilanosasociados a la lignina residual se eliminan y los grupos acetilo residualesse eliminan completamente. El comportamiento de los residuos de HexAdurante los procesos de blanqueo TCF y ECF son diferentes. Durante elblanqueo TCF, una pequeña proporción de los residuos MeGlcpA274

6. Conclusionesexistentes en la pasta se convierten en HexA mientras que durante elblanqueo ECF todos los HexA son eliminados por la acción del dióxidode cloro.10. El procedimiento biotecnológico basado en la utilización de un POM yla peroxidasa versátil (VP) del hongo Pleurotus eryngii muestra unagran eficacia para eliminar la lignina residual en pastas de celulosa. ElPOM en su forma oxidada es altamente selectivo para la deslignificacióny se reoxida completamente por la VP en tiempos muy cortos. De estamanera, es posible la sustitución de etapas de blanqueo que usan dióxidode cloro por tratamientos POM-VP-POM reox pudiendo utilizarse sistemasde este tipo en procesos industriales.11. El procedimiento biotecnológico basado en el uso de la lipoxigenasa delhongo Gaeumannomyces graminis muestra una gran eficacia paraeliminar los lípidos residuales responsables de los problemas de pitchdurante la producción de pastas de celulosa, especialmente en el caso depastas de lino. También se observa una mejora de algunas propiedadesde las pastas (especialmente en el caso de pastas de eucalipto) como esel aumento de la blancura y la disminución del número kappa.En conclusión, el estudio de la composición química de los cultivoslignocelulósicos utilizados como materia prima para la fabricación de pastasde celulosa así como la evolución de sus principales componentes durante losprocesos de cocción y blanqueo, contribuye a optimizar su aprovechamientoindustrial mediante tecnologías menos contaminantes. Este conocimientocontribuirá a un aprovechamiento industrial más sostenible de estosmateriales lignocelulósicos así como al desarrollo de nuevas especies deinterés socioeconómico.275

7. AnexosANEXOSAnexo 1. Peso de pasta ideal (c/ 7,5% humedad) para la determinación delíndice kappa. Adaptado de un documento realizado por Armindo Gaspar de laUniversidad de Aveiro.IK peso ideal/g peso seco/g70,0 0,190 0,17665,0 0,204 0,18960,0 0,221 0,20555,0 0,242 0,22450,0 0,266 0,24645,0 0,295 0,27340,0 0,332 0,30835,0 0,380 0,35130,0 0,443 0,41025,0 0,531 0,49220,0 0,664 0,61519,0 0,699 0,64718,0 0,738 0,68317,0 0,781 0,72416,0 0,830 0,76915,0 0,886 0,82014,0 0,949 0,87913,0 1,022 0,94612,0 1,107 1,02511,0 1,208 1,11810,0 1,328 1,2309,0 1,476 1,3678,0 1,661 1,5387,0 1,898 1,7576,0 2,214 2,0505,0 2,657 2,4604,0 3,321 3,7053,0 4,428 4,1002,0 6,642 6,1501,0 13,284 12,300277

7. AnexosAnexo 2. Factores f de corrección del consumo de permanganato usado en ladeterminación del índice kappa ( Tappi 2006).f+ 0 1 2 3 4 5 6 7 8 930 0,958 0,960 0,962 0,964 0,966 0,968 0,970 0,973 0,975 0,99740 0,979 0,981 0,983 0,985 0,987 0,989 0,991 0,994 0,996 0,99850 1,000 1,002 1,004 1,006 1,099 1,011 1,013 1,015 1,017 1,01960 1,002 1,024 1,026 1,028 1,030 1,033 1,035 1,037 1,039 1,04270 1,004278

7. AnexosAnexo 3. Peso de pasta ideal (c/ 7,5% humedad) para la determinación de laviscosidad intrínseca. Adaptado de un documento realizado por Armindo Gasparde la Universidad de Aveiro.Volumen del frasco/mlViscosidad 56 58 611400 0,129 0,134 0,1411350 0,134 0,139 0,1461300 0,139 0,144 0,1521250 0,145 0,150 0,1581200 0,151 0,156 0,1651150 0,158 0,163 0,1721100 0,165 0,171 0,1791050 0,173 0,179 0,1881000 0,181 0,188 0,197950 0,191 0,198 0,208900 0,201 0,209 0,219850 0,213 0,221 0,232800 0,227 0,235 0,247750 0,242 0,250 0,263700 0,259 0,268 0,282279

7. AnexosAnexo 4. Valores del producto []C para diferentes valores de rel(Scandinavian Pulp Paper and Board Committee 1994).0,00 0,01 0,02 0,03 0,04 0,05 0,06 0,07 0,08 0,091,0 0,000 0,010 0,020 0,030 0,040 0,049 0,059 0,069 0,078 0,0881,1 0,097 0,107 0,116 0,125 0,134 0,144 0,153 0,162 1,171 0,1801,2 0,189 0,198 0,207 0,216 0,224 0,233 0,242 0,250 0,259 0,2681,3 0,276 0,285 0,293 0,302 0,310 0,318 0,326 0,335 0,343 0,3511,4 0,359 0,367 0,375 0,383 0,391 0,399 0,407 0,415 0,423 0,4311,5 0,438 0,446 0,454 0,462 0,469 0,477 0,484 0,492 0,499 0,5071,6 0,514 0,522 0,529 0,537 0,544 0,551 0,558 0,566 0,573 0,5801,7 0,587 0,594 0,601 0,608 0,615 0,622 0,629 0,636 0,643 0,6501,8 0,657 0,664 0,671 0,678 0,684 0,691 0,698 0,705 0,711 0,7181,9 0,725 0,731 0,738 0,744 0,751 0,757 0,764 0,770 0,777 0,7832,0 0,790 0,796 0,802 0,809 0,815 0,821 0,827 0,834 0,840 0,8462,1 0,852 0,858 0,865 0,871 0,877 0,883 0,889 0,895 0,901 0,9072,2 0,913 0,919 0,925 0,931 0,937 0,943 0,949 0,954 0,960 0,9662,3 0,972 0,978 0,983 0,989 0,995 1,001 1,006 1,012 1,018 1,0232,4 1,029 1,035 1,040 1,046 1,051 1,057 1,062 1,068 1,073 1,0792,5 1,084 1,090 1,095 1,101 1,106 1,111 1,117 1,122 1,127 1,1332,6 1,138 1,143 1,149 1,154 1,159 1,164 1,170 1,175 1,180 1,1852,7 1,190 1,196 1,201 1,206 1,211 1,216 1,221 1,226 1,231 1,2362,8 1,241 1,246 1,251 1,256 1,261 1,266 1,271 1,276 1,281 1,2862,9 1,291 1,296 1,301 1,306 1,310 1,316 1,320 1,325 1,330 1,3353,0 1,339 1,344 1,349 1,354 1,358 1,363 1,368 1,373 1,377 1,3823,1 1,387 1,391 1,396 1,401 1,405 1,410 1,414 1,419 1,424 1,4283,2 1,433 1,437 1,442 1,446 1,451 1,455 1,460 1,464 1,469 1,4733,3 1,478 1,482 1,487 1,491 1,496 1,500 1,504 1,509 1,513 1,5173,4 1,522 1,526 1,531 1,535 1,539 1,544 1,548 1,552 1,556 1,5613,5 1,565 1,569 1,573 1,578 1,582 1,586 1,590 1,595 1,599 1,6033,6 1,607 1,611 1,615 1,620 1,624 1,628 1,632 1,636 1,640 1,6443,7 1,648 1,653 1,657 1,661 1,665 1,669 1,673 1,677 1,681 1,6853,8 1,689 1,693 1,697 1,701 1,705 1,709 1,713 1,717 1,721 1,7253,9 1,729 1,732 1,736 1,740 1,744 1,748 1,752 1,756 1,760 1,7644,0 1,767 1,771 1,775 1,779 1,783 1,787 1,790 1,794 1,798 1,8024,1 1,806 1,809 1,813 1,817 1,821 1,824 1,828 1,832 1,836 1,8394,2 1,843 1,847 1,851 1,854 1,858 1,862 1,865 1,869 1,873 1,8764,3 1,880 1,884 1,887 1,891 1,894 1,898 1,902 1,905 1,909 1,9124,4 1,916 1,920 1,923 1,927 1,930 1,934 1,937 1,941 1,944 1,948280

7. Anexos0,00 0,01 0,02 0,03 0,04 0,05 0,06 0,07 0,08 0,094,5 1,952 1,955 1,959 1,962 1,966 1,969 1,973 1,976 1,979 1,9834,6 1,986 1,990 1,993 1,997 2,000 2,004 2,007 2,010 2,014 2,0174,7 2,021 2,024 2,028 2,031 2,034 2,038 2,041 2,044 2,048 2,0514,8 2,054 2,058 2,061 2,064 2,068 2,071 2,074 2,078 2,081 2,0844,9 2,088 2,091 2,094 2,098 2,101 2,104 2,107 2,111 2,114 2,1175,0 2,120 2,124 2,127 2,130 2,133 2,137 2,140 2,143 2,146 2,1495,1 2,153 2,156 2,159 2,162 2,165 2,168 2,172 2,175 2,178 2,1815,2 2,184 2,187 2,191 2,194 2,197 2,200 2,203 2,206 2,209 2,2125,3 2,215 2,219 2,222 2,225 2,228 2,231 2,234 2,237 2,240 2,2435,4 2,246 2,249 2,252 2,255 2,258 2,261 2,264 2,267 2,270 2,2735,5 2,276 2,280 2,283 2,286 2,288 2,291 2,294 2,297 2,300 2,3035,6 2,306 2,309 2,312 2,315 2,318 2,321 2,324 2,327 2,330 2,3335,7 2,336 2,339 2,342 2,345 2,347 2,350 2,353 2,356 2,359 2,3625,8 2,365 2,368 2,371 2,374 2,376 2,379 2,382 2,385 2,388 2,3915,9 2,394 2,396 2,399 2,402 2,405 2,408 2,411 2,413 2,416 2,4196,0 2,422 2,425 2,427 2,430 2,433 2,436 2,439 2,441 2,444 2,4476,1 2,450 2,452 2,455 2,458 2,461 2,463 2,466 2,469 2,472 2,4756,2 2,477 2,480 2,783 2,485 2,488 2,491 2,494 2,496 2,499 2,5026,3 2,504 2,507 2,510 2,512 2,515 2,518 2,521 2,523 2,526 2,5296,4 2,531 2,534 2,537 2,539 2,542 2,545 2,547 2,550 2,552 2,5556,5 2,558 2,560 2,563 2,566 2,568 2,571 2,573 2,576 2,579 2,5816,6 2,584 2,587 2,589 2,592 2,594 2,597 2,599 2,602 2,605 2,6076,7 2,610 2,612 2,615 2,617 2,620 2,623 2,625 2,628 2,630 2,6336,8 2,635 2,638 2,640 2,643 2,645 2,648 2,651 2,653 2,656 2,6596,9 2,661 2,663 2,666 2,668 2,671 2,673 2,676 2,678 2,681 2,6837,0 2,686 2,688 2,690 2,693 2,695 2,698 2,700 2,703 2,705 2,7087,1 2,710 2,713 2,715 2,718 2,720 2,722 2,725 2,727 2,730 2,7327,2 2,735 2,737 2,739 2,742 2,744 2,747 2,749 2,752 2,754 2,7567,3 2,758 2,761 2,764 2,766 2,768 2,771 2,773 2,775 2,778 2,7807,4 2,783 2,785 2,787 2,790 2,792 2,794 2,797 2,799 2,801 2,8047,5 2,806 2,809 2,811 2,813 2,816 2,818 2,820 2,823 2,825 2,8277,6 2,829 2,832 2,834 2,836 2,839 2,841 2,843 2,846 2,848 2,8507,7 2,853 2,855 2,857 2,859 2,862 2,864 2,866 2,869 2,871 2,8737,8 2,875 2,878 2,880 2,882 2,885 2,887 2,889 2,891 2,894 2,8967,9 2,898 2,900 2,903 2,905 2,907 2,909 2,911 2,914 2,916 2,918281

7. Anexos0,00 0,01 0,02 0,03 0,04 0,05 0,06 0,07 0,08 0,098,0 2,920 2,923 2,295 2,927 2,929 2,932 2,934 2,936 2,938 2,9408,1 2,943 2,945 2,947 2,949 2,951 2,954 2,956 2,958 2,960 2,9628,2 2,964 2,967 2,969 2,971 2,973 2,975 2,978 2,980 2,982 2,9848,3 2,986 2,988 2,991 2,993 2,995 2,997 2,999 3,001 3,003 3,0068,4 3,008 3,010 3,012 3,014 3,016 3,018 3,020 3,023 3,025 3,0278,5 3,029 3,031 3,033 3,035 3,037 3,040 3,042 3,044 3,046 3,0488,6 3,050 3,052 3,054 3,056 3,058 3,061 3,063 3,065 3,067 3,0698,7 3,071 3,073 3,075 3,077 3,079 3,081 3,083 3,085 3,087 3,0908,8 3,092 3,094 3,096 3,098 3,100 3,102 3,104 3,106 3,108 3,1108,9 3,112 3,114 3,116 3,118 3,120 3,122 3,124 3,126 3,128 3,1309,0 3,132 3,134 3,136 3,138 3,340 3,142 3,144 3,147 3,149 3,1519,1 3,153 3,155 3,157 3,159 3,161 3,163 3,165 3,166 3,168 3,1709,2 3,172 3,174 3,176 3,178 3,180 3,182 3,184 3,186 3,188 3,1909,3 3,192 3,194 3,196 3,198 3,200 3,202 3,204 3,206 3,208 3,2109,4 3,212 3,214 3,216 3,218 3,220 3,222 3,223 3,225 3,227 3,2299,5 3,231 3,233 3,265 3,237 3,239 3,241 3,242 3,245 3,247 3,2499,6 3,250 3,252 3,254 3,256 3,258 3,260 3,262 3,264 3,266 3,2689,7 3,270 3,271 3,273 3,175 3,277 3,273 3,281 3,283 3,285 3,8879,8 3,288 3,290 3,292 3,294 3,296 3,298 3,300 3,302 3,303 3,3059,9 3,307 3,309 3,311 3,313 3,315 3,316 3,318 3,320 3,322 3,32410 3,326 3,344 3,363 3,381 3,399 3,416 3,434 4,452 3,469 3,48711 3,504 3,521 3,538 3,554 3,571 3,588 3,604 3,620 3,636 3,65312 3,669 3,684 3,700 3,716 3,731 3,747 3,762 3,777 3,792 3,80713 3,822 3,837 3,852 3,866 3,881 3,895 3,910 3,924 3,938 3,95214 3,966 3,980 3,994 4,008 4,021 4,035 4,048 4,062 4,075 4,08815 4,101 4,115 4,128 4,141 4,153 4,166 4,179 4,192 4,204 4,21716 4,229 4,242 4,254 4,266 4,279 4,291 4,303 4,315 4,327 4,33917 4,351 4,362 4,374 4,386 4,397 4,409 4,420 4,432 4,443 4,45518 4,466 4,477 4,488 4,499 4,510 4,521 4,532 4,543 4,554 4,56519 4,576 4,586 4,597 4,608 4,618 4,629 4,639 4,650 4,660 4,670282