Desarrollo de pretratamientos enzimáticos de materiales lignocelulósicos para la obtención de bioetanol de segunda generación.
Abstract
La presente Tesis aborda el estudio de pretratamientos biológicos destinados a romper la barrera de lignina y mejorar la sacarificación de los materiales lignocelulósicos utilizados en la obtención de bioetanol de segunda generación. Los pretratamientos estudiados en esta Tesis están basados en el uso del sistema enzimático lacasa-mediador y han demostrado ser eficaces en degradar/modificar la estructura del polímero de lignina que envuelve y protege a los carbohidratos. Las modificaciones en la estructura de la lignina permiten que los carbohidratos queden más expuestos a las enzimas celulasas, lo que se traduce en una mejora significativa en la hidrólisis de los mismos. Por otro lado, esta Tesis también plantea el estudio de las interacciones que tienen lugar entre la lignina y las celulasas, y que afectan al proceso de hidrólisis de la celulosa. Estos estudios tienen como objetivo un aprovechamiento más racional y eficaz de los materiales lignocelulósicos en la producción de biocombustibles de segunda generación (bioetanol). En esta Tesis se han evaluado dos sistemas lacasa-mediador. El primero está constituido por una lacasa de alto potencial redox, obtenida del hongo basidiomiceto Pycnoporus cinnabarinus, y el mediador redox sintético, 1-hidroxibenzotriazol (HBT). El segundo, incluye una lacasa comercial obtenida del hongo ascomiceto Myceliophthora thermophila y el mediador natural siringato de metilo (MeS). Los materiales lignocelulósicos utilizados en los pretratamientos enzimáticos con el sistema lacasa-mediador incluyeron: i) residuos agrícolas de paja de trigo, el bagazo y la paja de la caña de azúcar y ii) madera de paulownia (Paulownia fortunei). Para el estudio de las interacciones lignina-celulasa se utilizaron ligninas aisladas de eucalipto (Eucalyptus globulus), paja de trigo y pícea (Picea abies) . Los pretratamientos de materiales lignocelulósicos con lacasa de P. cinnabarinus en presencia/ausencia de HBT, se realizaron sobre dos materias primas, los residuos agrícolas de paja de trigo y de caña de azúcar, con el objeto de mejorar el rendimiento de sacarificación enzimática y obtención de bioetanol de ambas materias. Primeramente se realizaron experimentos del tratamiento sobre la paja de trigo en una secuencia que incluía un solo ciclo enzimático seguido de una extracción alcalina con peróxido de hidrógeno. Tras la secuencia se determinó el contenido en lignina (lignina Klason) y se evaluó el efecto que esta eliminación/modificación de lignina producía sobre la sacarificación enzimática (con celulasas). Este pretratamiento produjo un descenso en el contenido en lignina del 45% respecto a la materia prima y una mejora en la obtención de glucosa del 60% tras 72 h de tratamiento con celulasas. También se analizó la modificación de la estructura de lignina con dichos tratamientos mediante resonancia magnética nuclear bidimensional (2DRMN). Estos estudios revelaron un descenso en los enlaces β-O-4´y β-5´ así como en las unidades de lignina p-hidroxifenilo (H), guayacilo (G) y siringilo (S), y una disminución más moderada de los ácidos p-cumárico y ferúlico, sin variación sustancial en la unidad de tricina. Con el objeto de estudiar la eficacia del sistema lacasa-mediador sobre otras materias primas, se estudió la aplicación del mismo sobre los residuos de la industria de la caña de azúcar (bagazo y paja), en este caso utilizando una secuencia de 4 ciclos con dosis menores de enzima y mediador por ciclo. Con esta secuencia se obtuvo un descenso en el contenido en lignina del 27% y 31% para el bagazo y paja de caña de azúcar respectivamente, y una mejora del 39% y 46% en el rendimiento de glucosa, en la materia pretratada de bagazo y paja, en comparación con el control, tras las 72 h de tratamiento con celulasas. El análisis 2D-RMN reveló notables cambios en la estructura de la estructura de la lignina, observándose en ambos materiales un descenso significativo en el número los enlaces y unidades de lignina. La eficacia del sistema lacasa-mediador volvió a quedar demostrada con el pretratamiento realizado sobre madera de paulownia con la lacasa de M. thermophila y MeS. Dicho pretratamiento constó de una secuencia de 4 ciclos (cada uno de ellos incluía un tratamiento enzimático seguido de una extracción alcalina). Los mejores resultados se obtuvieron con 50 U·g-1 de enzima y 3% de MeS, consiguiendo un descenso en lignina del 24% respecto al control y un aumento en la liberación de glucosa del 40%. Los análisis de 2D-NMR de estos materiales revelaron un descenso en el número de enlaces y un aumento de las unidades oxidadas de siringilo y guayacilo, tras el pretratamiento. Por último, se analizaron los filtrados obtenidos durante los diferentes ciclos, encontrándose compuestos oxidados de lignina como vainillina, ácido vanílico, siringaldehído y ácido siríngico, que corroboran la naturaleza oxidativa del pretratamiento y la consecuente deslignificación de los materiales lignocelulósicos. Con el fin de demostrar el efecto negativo que tiene la presencia de lignina sobre las celulasas en la hidrólisis del material lignocelulósico, se aislaron las ligninas (milled wood lignin, MWL) de diferentes materias primas (eucalipto, paja de trigo y pícea), y se estudió su efecto en la adsorción y consecuente inhibición de las celulasas (tanto sobre el cóctel enzimático, como sobre la enzima purificada) mediante microbalanza de cristal de cuarzo y resonancia de plasmón superficial. Los resultados al aplicar las celulasas sobre films recubiertos de dichas ligninas demostraron que la enzima purificada muestra menor afinidad a la adsorción sobre el film con lignina y mayor reversibilidad de unión. También se observó que la afinidad más alta en la adsorción lignina-enzima se produce en la lignina de pícea (99% unidades G) mientras que la adsorción más baja se produjo en la de eucalipto (70% unidades S).
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DESARROLLO DE PRETRATAMIENTOS ENZIMÁTICOS DE MATERIALES LIGNOCELULÓSICOS PARA LA OBTENCIÓN DE BIOETANOL DE SEGUNDA GENERACIÓN Antonio Pereira González Sevilla, 2018
Desarrollodepretratamientos enzimáticosdematerialeslignocelulósicos paralaobtencióndebioetanolde segundageneración Memoriaquepresenta: AntonioPereiraGonzález paraoptaraltítulodeDoctoren CienciasQuímicasporlaUniversidad deSevilla
PcL Lacasa de Pycnoporus cinnabarinus QCM Microbalanza de cristal de cuarzo (“Quarz Crystal Microbalance”) S Unidad siringilpropano (o siringilo) S´ Unidad siringilpropano oxidada SA Siringaldehído SPR Resonancia de plasmón superficial (“Surface Plasmon Resonance”) T20 Tween 20 U Unidad de actividad enzimática
ÍNDICE RESUMEN ………………………………………………………………………………………… I I.INTRODUCCIÓN …………………………………………………………………………….. 1 I.1. CULTIVOS LIGNOCELULÓSICOS …………………………………………………. 3 I.1.1. Cultivos forestales ………………………………………………………………. 3 I.1.2. Cultivos agrícolas ………………………………………………………………… 4 I.2. ESTRUCTURA Y COMPOSICIÓN DE LA LIGNOCELULOSA …………….. 5 I.2.1. Celulosa ………………………………………………………………………………. 6 I.2.2. Hemicelulosas …………………………………………………………………….. 8 I.2.3. Lignina ………………………………………………………………………………… 10 I.2.4. Compuestos minoritarios ……………………………………………………. 17 I.3. BIORREFINERÍAS DE LA LIGNOCELULOSA …………………………………… 18 I.3.1. Producción de pasta de papel …………………………………………….. 20 I.3.2. Producción de biocombustibles de segunda generación …….. 21 I.3.2.1. Pretratamientos …………………………………………………………… 24 (a) Pretratamientos físicos ………………………………………………….. 25 (b) Pretratamientos químicos ……………………………………………… 25 (c) Pretratamientos físico-químicos …………………………………….. 2ϳ (d) Pretratamientos biológicos………………………………………..….Ϯϵ I.3.2.2. Hidrólisis ………………………………………………………………………. 30 I.3.2.3. Fermentación ………………………………………………………………… 31 I.4. BIODEGRADACIÓN ENZIMÁTICA DE LA LIGNINA ……………………….. 31 I.5. INFLUENCIA DE LA LIGNINA EN LA SACARIFICACIÓN DEL MATERIAL LIGNOCELULÓSICO………………………………………………………………………… 34 II. OBJETIVOS ………………………………………………………………………………….. 39 III. RESULTADOS GENERALES Y DISCUSIÓN ………………………………………… 43 III.1. PRETRATAMIENTOS ENZIMÁTICOS CON EL SISTEMA LACASAMEDIADOR …………………………………………………………………………………….. 45
III.1.1. Pretratamiento enzimático de paja de trigo con lacasa de Pycnoporus cinnabarinus y HBT ………………………………………..…. 45 III.1.2. Pretratamiento enzimático de bagazo y paja de caña de azúcar con lacasa de Pycnoporus cinnabarinus y HBT …………….. 48 III.1.3. Pretratamiento enzimático de Pawlonia fortunei con lacasa de Myceliophthora thermophila y siringato de metilo……….. 51 III.2. INTERACCIONES LIGNINA-CELULASAS …………………………………….. 55 IV. REFERENCIAS …………………………………………………………………………….. 61 V. PUBLICACIONES CIENTÍFICAS ………………………………………………………. 75 Publicación 1: Laccase-mediator pretreatment of wheat straw degrades lignin and improves saccharification ………………………………. 77 Publicación 2: Delignification and saccharification enhancement of sugarcane byproducts by a laccase-based pretreatment …....…… 93 Publicación 3: A commercial laccase -mediator system to delignify and improve saccharification of the fast-growing Paulownia fortunei.….. 105 Publicación 4: Lignin films from spruce, eucalyptus, and wheat straw studied with electroacoustic and optical sensors: effect of composition and electrostatic screening on enzyme binding….…………………………… 131 VI. CONCLUSIONES ………………………………………………………………………….. 145
RESUMEN La presente Tesis aborda el estudio de pretratamientos biológicos destinados a romper la barrera de lignina y mejorar la sacarificación de los materiales lignocelulósicos utilizados en la obtención de bioetanol de segunda generación. Los pretratamientos estudiados en esta Tesis están basados en el uso del sistema enzimático lacasa-mediador y han demostrado ser eficaces en degradar/modificar la estructura del polímero de lignina que envuelve y protege a los carbohidratos. Las modificaciones en la estructura de la lignina permiten que los carbohidratos queden más expuestos a las enzimas celulasas, lo que se traduce en una mejora significativa en la hidrólisis de los mismos. Por otro lado, esta Tesis también plantea el estudio de las interacciones que tienen lugar entre la lignina y las celulasas, y que afectan al proceso de hidrólisis de la celulosa. Estos estudios tienen como objetivo un aprovechamiento más racional y eficaz de los materiales lignocelulósicos en la producción de biocombustibles de segunda generación (bioetanol). En esta Tesis se han evaluado dos sistemas lacasa-mediador. El primero está constituido por una lacasa de alto potencial redox, obtenida del hongo basidiomiceto Pycnoporus cinnabarinus, y el mediador redox sintético, 1-hidroxibenzotriazol (HBT). El segundo, incluye una lacasa comercial obtenida del hongo ascomiceto Myceliophthora thermophila y el mediador natural siringato de metilo (MeS). Los materiales lignocelulósicos utilizados en los pretratamientos enzimáticos con el sistema lacasa-mediador incluyeron: i) residuos agrícolas de paja de trigo, el bagazo y la paja de la caña de azúcar y ii) madera de paulownia (Paulownia fortunei). Para el estudio de las interacciones lignina-celulasa se utilizaron ligninas aisladas de eucalipto (Eucalyptus globulus), paja de trigo y pícea (Picea abies) . Los pretratamientos de materiales lignocelulósicos con lacasa de P. cinnabarinus en presencia/ausencia de HBT, se realizaron sobre dos materias primas, los residuos agrícolas de paja de trigo y de caña de azúcar, con el objeto de mejorar el rendimiento de sacarificación
enzimática y obtención de bioetanol de ambas materias. Primeramente se realizaron experimentos del tratamiento sobre la paja de trigo en una secuencia que incluía un solo ciclo enzimático seguido de una extracción alcalina con peróxido de hidrógeno. Tras la secuencia se determinó el contenido en lignina (lignina Klason) y se evaluó el efecto que esta eliminación/modificación de lignina producía sobre la sacarificación enzimática (con celulasas). Este pretratamiento produjo un descenso en el contenido en lignina del 45% respecto a la materia prima y una mejora en la obtención de glucosa del 60% tras 72 h de tratamiento con celulasas. También se analizó la modificación de la estructura de lignina con dichos tratamientos mediante resonancia magnética nuclear bidimensional (2D- RMN). Estos estudios revelaron un descenso ĞŶůŽƐĞŶůĂĐĞƐɴ-O-ϰDzLJ ɴ-5´ así como en las unidades de lignina p-hidroxifenilo (H), guayacilo (G) y siringilo (S), y una disminución más moderada de los ácidos p-cumárico y ferúlico, sin variación sustancial en la unidad de tricina. Con el objeto de estudiar la eficacia del sistema lacasa-mediador sobre otras materias primas, se estudió la aplicación del mismo sobre los residuos de la industria de la caña de azúcar (bagazo y paja), en este caso utilizando una secuencia de 4 ciclos con dosis menores de enzima y mediador por ciclo. Con esta secuencia se obtuvo un descenso en el contenido en lignina del 27% y 31% para el bagazo y paja de caña de azúcar respectivamente, y una mejora del 39% y 46% en el rendimiento de glucosa, en la materia pretratada de bagazo y paja, en comparación con el control, tras las 72 h de tratamiento con celulasas. El análisis 2D-RMN reveló notables cambios en la estructura de la estructura de la lignina, observándose en ambos materiales un descenso significativo en el número los enlaces y unidades de lignina. La eficacia del sistema lacasa-mediador volvió a quedar demostrada con el pretratamiento realizado sobre madera de paulownia con la lacasa de M. thermophila y MeS. Dicho pretratamiento constó de una secuencia de 4 ciclos (cada uno de ellos incluía un tratamiento enzimático seguido de una extracción alcalina). Los mejores resultados se obtuvieron con 50 U·g-1 de enzima y 3% de MeS, consiguiendo un descenso en lignina del 24% respecto al control y un aumento en la liberación de glucosa del 40%. Los
análisis de 2D-NMR de estos materiales revelaron un descenso en el número de enlaces y un aumento de las unidades oxidadas de siringilo y guayacilo, tras el pretratamiento. Por último, se analizaron los filtrados obtenidos durante los diferentes ciclos, encontrándose compuestos oxidados de lignina como vainillina, ácido vanílico, siringaldehído y ácido siríngico, que corroboran la naturaleza oxidativa del pretratamiento y la consecuente deslignificación de los materiales lignocelulósicos. Con el fin de demostrar el efecto negativo que tiene la presencia de lignina sobre las celulasas en la hidrólisis del material lignocelulósico, se aislaron las ligninas (milled wood lignin, MWL) de diferentes materias primas (eucalipto, paja de trigo y pícea), y se estudió su efecto en la adsorción y consecuente inhibición de las celulasas (tanto sobre el cóctel enzimático, como sobre la enzima purificada) mediante microbalanza de cristal de cuarzo y resonancia de plasmón superficial. Los resultados al aplicar las celulasas sobre films recubiertos de dichas ligninas demostraron que la enzima purificada muestra menor afinidad a la adsorción sobre el film con lignina y mayor reversibilidad de unión. También se observó que la afinidad más alta en la adsorción lignina-enzima se produce en la lignina de pícea (99% unidades G) mientras que la adsorción más baja se produjo en la de eucalipto (70% unidades S).
I. INTRODUCCIÓN Pycnoporus cinnabarinus
Introducción I I.1.CULTIVOSLIGNOCELULÓSICOS Labiomasavegetalrepresentalaprincipalfuentedemateriales renovablesenlaTierra.Dichabiomasaseencuentraprincipalmenteenlas paredescelularesdelasplantasyenglobaespeciestantoagrícolascomo forestalesdegraninterésindustrialtantoparalaproduccióndepastade celulosa,cuyaprincipalfuentesonlasfibrasmadereras,comode biocombustiblesqueseobtienenprincipalmenteapartirdecultivosde origennomadererodebidoasugrandisponibilidadybajocoste.Además, losresiduosgeneradosporestoscultivospresentantambiéngraninterés comomateriaprimaparalaobtencióndecelulosayotrosproductos. I.1.1.Cultivosforestales Estánconstituidospordiferentesespeciesdeconíferasyfrondosas.Las coníferasseutilizanprincipalmenteparalaproduccióndepastadepapel debidoalauniformidaddelamadera,laaltaresistenciamecánicayla presenciadefibraslargas(entre3y5mm)(GarcíaHortal,2007).Lapícea (Figura1)yelpinosonlasespeciesdeconíferasmásutilizadasenla industriapapelera. Figura1.Cultivosforestalesrepresentativosdeconíferascomolapícea(A)yfrondosas comoeleucalipto(B). AB 3
IIntroducción enzimashemicelulasas,queeliminantantolascadenaslateralescomola cadenaprincipalaleatoriamente,paraliberaroligosacáridosqueserán degradadosenazúcaressimples(AbdelͲHamidetal.,2013). I.2.3.Lignina Laligninaes,traslacelulosa,elpolímeromásabundanteenlasuperficie terrestre,seencuentraenlamayoríadeplantasterrestresenlasque representaentreel25Ͳ33%delabiomasasecaenmaderasdeconíferasy entreel18Ͳ34%enlamaderadefrondosas(Boerjanetal.,2003; Ragauskas,2014).Laligninarealizadiversasfuncionesentrelasque destacansupapeleneltransporteinternodeagua,metabolitosy nutrientesyproporcionaunasuperficiehidrófobaalsistemavasculardela planta.Tambiéntienefuncióncomoaglomerantedelasfibrasdecelulosa yhemicelulosasdebidoasucarácterhidrófobo,proporcionandorigidezy resistenciaalaparedcelular,asícomoprotecciónanteagentespatógenos (SarkanenyLudwig,1971;Chandraetal.,2007). Estructuralmente,laligninaesunheteropolímeroaromático,muy ramificadoyamorfo.Estáconstituidoporunidadesdefenilpropanocon diferentespatronesdesustituciónunidospordiferentesenlaces,que varíanconsiderablementeentrelasdiferentesespeciesvegetales(Ralph etal.,2004;Boerjanetal.,2003;Vanholmeetal.,2010). Laligninasesintetizaapartirdelaminoácidofenilalaninamediantelaruta delácidocinámico(Figura7)(FreudenbergyNeish,1968;Adler,1977; Boerjanetal.,2003;Higuchi,2012).Unaseriedeenzimassonlas encargadasdelaconversióndelLͲfenilalaninaenlosalcoholespͲ cumarílico,coniferílicoysinapílicoqueactúancomoprecursoreso monómerosdelalignina.EstosalcoholespͲhidroxicinamílicos,también conocidoscomomonolignoles,solosediferencianestructuralmenteenel númerodemetoxilospresentessusanillosaromáticos(Figura7). ϭϬ
Introducción I Figura 7. Ruta ďŝŽƐŝŶƚĠƟĐĂ de la lignina (adaptada de Boerjan et al., 2003). Existen ĚŝƐƟŶƚĂƐ rutas, pero la ruta de ĐŽůŽƌ azul es la más ĨĂǀŽƌĞĐŝĚĂ En la ruta intervienen ŽŶĐĞ enzimas, que en orden de ĂĐƚƵĂĐŝſŶ son las siguientes: amonio liasa (PAL), Đinamato-4-hidroxilasa (C4H), 4-Đumarato CoA ligasa (4CL), ĐĂĨĞiĐo O-ŵĞƟůƚƌĂŶƐĨĞƌasa (COMT), Đŝnamoil CoA-reĚƵĐƚĂƐĂ (CCR), ĐĂĨeoil CoA-3-O-meƟůƚranferasa (CCoAOMT), Đinamil alĐohol deshidrogenasa (CAD), ferulato-5-hidroxilasa (F5H), sinapil ĂůĐŽŚŽů deshidrogenasa (SAD), p-Đumarato-3-hidroxilasa (C3H) y ĐĂīĞŽLJů shikimate esterase (CSE). 11 OH OH OMe OH OH OH OH OH OH OH OMe HO COMT F5H COMT COMT CCoAOMT F5H COMT CCoAOMT C4H/C3H COMT F5H COMT COMT F5H COMT C4H PAL 4CL 4CL 4CL 4CL 4CL CCR CCR CCR CCR CCR Fenilalanina Ácido cinámico Ácido p-cumárico p-Cumaroil-CoA p-Cumaraldehído Alcoholp-cumarílico Alcohol cafeílico Alcohol coniferílico Alcohol 5-hidroxiconiferílico Alcohol sinapílico Cafeilaldehído Coniferaldehído 5-Hidroxiconiferaldehído Sinapaldehído Cafeoil-CoA Feruloil-CoA 5-Hidroxiferuloil-CoA Ácido sinápico Sinapoil-CoA Ácido cafeico Ácido ferúlico Ácido 5-hidroxiferúlico Peroxidasa/Lacasa Peroxidasa/Lacasa Peroxidasa/Lacasa LIGNINA C3H Ácido p-cumaroil siquímico OH O O O OH HO HO HCT Ácido cafeoil siquímico OH OO O OH HO HO HO HCT CSE C3H Ácido p-cumaroil siquímico OH O O O OH HO HO HCT Ácido cafeoil siquímico OH O O O OH HO HO HO HCT CSE O HO NH2 O HO O HO OH CoA O S OH O H OH SADCAD OH OH O HO OH OH O HO OH OMe O HO OH OMe HO O HO OH OMe MeO O OH OH O OH OMe O OH OMe HO O OH OMe MeO CoA SCoA SCoA SCoA S O OH OH O OH OMe O OH OMe HO O OH OMe MeO HHHH OMeMeO SADCAD SADCAD SADCAD SADCAD
IIntroducción Estos monolignoles se incorporan a la lignina y dan lugar a las distintas unidades de phidroxifenilo (H), guayacilo (G) y siringilo (S) (Figura 8). La composición de la lignina en términos H:G:S varía ampliamente en la biomasa vegetal dependiendo de la especies. En términos generales, la lignina de plantas herbáceas está constituida por las tresunidades monoméricas G, S y H, la lignina de frondosas contiene unidades G y S en distinta proporción y la lignina de coníferas está básicamente compuesta por unidades G (Abdel Hamid et al., 2013). La composición de la lignina también varía con la edad de la planta (Freudenberg y Neish, 1968; Rencoret et al., 2010), el lugar de la paredcelular o lámina media donde se sintetice (Fukushima y Terashima,1991; Christiernin et al., 2005) y el tejido de la misma (Bland 1966; Hardell et al., 1980a,b; Lourenço et al., 2016). Figura 8. Unidades phidroxifenilo (H), guayacilo (G) y siringilo (S) de la lignina, que se forman tras la polimerización de losrespectivos monolignoles, alcohol pcumarílico, coniferílico y sinapílico (adaptado de Ralph et al., 2004). OH OH O OMeMeO OH OH OMeMeO OH OH OMe O OMe O SGH p-Hidroxifenilo Guayacilo Siringilo alc. p-cumarílico alc. coniferílico alc. sinapílico
Introducción I LasunidadesSdeligninatienendosgruposmetoxilosenlasposicionesCͲ 3yCͲ5porloquenopuedenestablecerenlacescondensadoscarbonoͲ carbonoenelanillo.Principalmenteseunenaotrasunidadesmediante enlacesdetipoéteratravésdelgrupohidroxilodesuCͲ4y,porlotanto, formanligninasconunaestructuralinealymásfácildedegradar.Porel contrario,lasunidadesHtienenlibreslosCͲ3yCͲ5,yaquenoposeen gruposmetoxilos,porloqueconsecuentementeestasunidadespueden formarenlacesCͲCenestasposicionesyformaránligninasconmayor gradodecondensación.LasunidadesGseencuentranenuntérmino intermedioyaquetienenocupadalaposiciónCͲ3porungrupometoxiloy laCͲ5libreydisponibleparalaformacióndeenlacescarbonoͲcarbono, porloquelasligninasricasenunidadesGseránmásdifícilesdedegradar queaquellasenriquecidasenunidadesS(delRíoetal.,2005). Ademásdelostresmonolignolestradicionales,sehandescritootros compuestosfenólicosquetambiénactúancomomonómerosdelalignina participandoenreaccionesdeacoplamientodurantelalignificación.Entre ellosseencuentranlosderivadosaciladosdeloscorrespondientes alcoholespͲhidroxicinamílicosconacetatos,pͲcumaratos,pͲ hidroxibenzoatos,ferulatos,benzoatosyvainillatos(Figura9A)(delRíoet al.,2007,2008;Martínezetal.,2008;Rencoretetal.,2013,Karlenetal. 2017),compuestosintermediariosdelarutabiosintéticadeligninacomo elaldehídocinamílico,ácidoferúlico,alcoholcafeílicoyalcohol5Ͳ hidroxiconiferílico(Figura9B)(Kimetal.,2003;Lepléetal.,2007;Chenet al.,2013)einclusocompuestosfenólicosprocedentesdeotrasrutas biosintéticas(flavonoidesyestilbenoides).Estosúltimoshansido descubiertosrecientemente,elprimerodeellosfuelaflavonatricina, presenteenlasligninasdegramíneasyotrasmonocotiledóneas(delRíoet al.,2012),yrecientementesehandescritoloshidroxiestilbenos piceatanol,isorhapontigeninayresveratrol(Figura9C),presentesenlas ligninasdelosendocarposdefrutosdepalmas(delRíoetal.,2017; Rencoretetal.,2018). ϭ3
IIntroducción Figura 9. Otros monómeros encontrados en el polímero de lignina. A) alcoholes p hidroxicinamílicos acilados, B) compuestos intermediarios de la ruta biosintética de la lignina, C) compuestos que proceden de otras rutas biosintéticas (flavonoides y estilbenoides). La lignificación comienza con la deshidrogenación enzimática (mediante peroxidasas y/o lacasas) de los monolignoles, que da lugar a la formación de radicales libres tipo fenoxilo, estabilizados por resonancia (Figura 10). Figura 10. Deshidrogenación enzimática del alcohol coniferílico y formas resonantes de radical fenoxilo (adaptado de Adler, 1977). Acontinuación, tiene lugar el acoplamiento de los radicales entre sí y con el polímero creciente de lignina mediante diversos tipos de enlaces. Las uniones que se producen se pueden clasificar en dosgrandesgrupos, enlaces de tipo éter y uniones de tipo carbono carbono (Figura 11). Las OH O OMe OH O OMe OH O OMe OH O OMe OH OH OMe 1 2 3 4 5 6 αβ γ Peroxidasa/ lacasa - (e + H+) OH O OMe O O OH OMe O O OH OMe HO coniferil/sinapil acetato coniferil/sinapil p-hidroxibenzoato O O OH OMe HO coniferil/sinapil p-cumarato coniferil/sinapil ferulato HO OH OH alc. cafeílico alc. 5-hidroxiconiferílico HO OH OMe HO OH OMe O H coniferaldehído/ sinapaldehído A) B) O O OH OMe HO OMe O O OH OMe coniferil/sinapil benzoato coniferil/sinapil vainillato O O OH OMe HO OMe C) OH OHO O OMe OMe OH tricina OH HO R OH R=OH piceatanol R=OMe isorhapontigenina R=H resveratrol
Introducción I uniones tipo éter incluyen los enlaces O4 y 4 O5, siendo el enlace O 4 el más común en la lignina. Las uniones carbono carbono, también conocidas como enlaces condensados, incluyen los enlaces resinol, formados por la unión de dos cadenas alifáticas, los enlaces 1, al unirse un carbonodeuna cadena alifática con un carbono de un anillo bencénico de otra unidad, formando una estructura de tipo espirodienona (Zhang y Gellerstedt, 2001), enlaces 5 fenilcumaranos, y enlaces 5 5, formados por la unión de carbonos de anillos bencénicos, que se encuentra en forma de trímero, al incorporarse una terceraunidad mediante enlaces O4 / O4 dando lugar a una estructura de tipo dibenzodioxocina (Karhunen et al. 1995). La proporción de estos enlaces varía en las diferentes especies de plantas, siendo el O4 el más abundante. Figura 11.Principales subestructuras presentes en la lignina. A) O 4 alquil aril éter, B) 5 fenilcumarano, C) resinol, D) 5 5 dibenzodioxacina, E) 1 espirodienona, F) 1, J) 4 O 5 aril aril éter. El conocimiento en la estructura de la lignina ha avanzado enormemente en losúltimos añosgracias a los avances en las técnicas analíticas, A HO O HO OMe OMe O αβ4′ γ O HO OMeO OMe 5′ B α β γ O O OMe O O OMe C α β γ α′ β′ γ′ α′ α β γ1′ β′ γ′ 2′ 6′ 5′ E O O OH HO OMe O OMe OAr 5′ 5′′ αβ γ 4′ 4′′ D OH OMe MeO OO OOMe F HO HO OMe OMe O O α β γ 1′ O OMe OH OMe 5′4 J
I Introducción especialmente en la 2D-NMR. En la Figura 12 se muestra un modelo estructural de las ligninas de coníferas y de frondosas. Figura 12. Modelo del polímero de lignina en madera: A) conífera (pícea) (Brunow, 2005) y B) frondosa (álamo) (Boerjan et al., 2003). La lignina también se asocia a los polisacáridos mediante enlaces covalentes formando los llamados complejos macromoleculares lignina- 16 A) B)
Introducción I carbohidrato (Figura 13). Existen tres tipos de enlaceslignina carbohidrato, glucósidos de fenilo, ésteres del ácido 4 Ometilglucurónico con el C de la lignina y ésteres de bencilo (Fengel y Wegener,1983; Balakshin et al., 2011; del Río et al., 2016). Figura 13.Principales uniones lignina carbohidrato observadas en plantas: fenil glicósidos(PhGlc),lignina carbohidrato ester (Est) y bencil éteres (BE). I.2.4. Compuestos minoritarios Además de la celulosa, las hemicelulosas y la lignina,existen otros componentes que se presentan en menor proporción y entre los cuales se encuentran los extraíbles hidrofílicos ylipofílicos, proteínas, pectinas y compuestos inorgánicos (Sun,2010). Estos compuestos minoritarios no influyen en la estructura morfológica de la pared celular, pero desempeñan funciones vitales para la célula vegetal como la protección a insectos. Además son losresponsables del color y olor de la madera. Sin embargo, algunos de ellostienen un efectonegativo en la producción de la pasta de papel, como porejemplo la formación de depósitos de pitch en la fabricación de la pasta de celulosa producida por algunos extraíbles lipofílicos (Gutiérrez et al., 2001, 2006). Extraíbles hidrofílicos:engloban compuestos fenólicos libres de bajo peso molecular, que se pueden aislar del material lignocelulósico mediante extracciones con disolventes polares. Se incluyen dentro de este grupo precursores de lignina (ácidos phidroxicinamílicos y aldehídos p OMe O C 1 -carb PhGlc O O HO OMe OMe O αβ4′ γ OOMe OH OH O O Carb Est HO O O R OMe OMe O αβ4′ γ BE BE 1 : R=C6 Glc, Man,Gal C5 in Ara BE 2 : R=C2 or C3 in Xyl, Glc, Man, Gal, Ara
IIntroducción hidroxicinámicos),ácidosbencenocarboxílicosrelacionados(ácidopͲ hidroxibenzoico,vainíllicoysiríngico),aldehídosycetonasaromáticas(pͲ hidroxibenzaldehído,vainillinaysiringaldehídoypropioguayacona), lignanos(dilignolesycompuestosrelacionados),taninoshidrolizables (ésteresdeácidogálicoysusdímeros),taninosnohidrolizables(varias unidadesdeflavonoidescondensadas)yflavonoides(estructuraderivadas delanillodeflavona). Extraíbleslipofílicos:incluyenalcanos,alcoholesgrasos,aldehídos,ácidos grasos,esteroleslibresyconjugados,ácidosresínicos,ceras(ésteresde ácidograsoconalcoholesdecadenalarga)yglicéridos(ésteresdeácido grasoconglicerol).Sepuedenobtenerestosextraíbleslipófilosdel materiallignocelulósicocondisolventesapolares. I.3.BIORREFINERÍASDELALIGNOCELULOSA Elconceptodebiorrefineríadelalignocelulosasebasaenaprovechar íntegramenteestabiomasavegetalrenovableparaproducirbioenergíay biomaterialesmediantelaaplicacióndeprocesosfísicos,químicosy/o biológicos.Losprincipalesusosindustrialessonlaobtencióndecelulosa paralaproduccióndepastadepapelylaobtencióndebiocombustiblesde segundageneración(bioetanol),asícomolavalorizacióndelalignina,que sueleserunresiduoenlosprocesosanteriores. Elobjetivoactualdelabiorrefineríaesconseguirel“zerowaste”,este términohacereferenciaalusoeficientedetodoslosazúcaresycarbonos unidosenlabiomasalignocelulósica(Youngetal.,2010).LaFigura14 muestraelusoqueseledaalosdistintoscomponentesmayoritariosdel materiallignocelulósico. ϭϴ
Introducción I Figura14.Representaciónesquemáticadeusosdelacelulosa,hemicelulosaylignina enlaindustria. Losprincipalesusosindustrialesdelabiomasalignocelulósicasehan centradoprincipalmenteenlaobtencióndelacelulosaparalaproducción depastadepapelydebiocombustibles.Enestosprocesoslaligninaesun residuoquesequemaparagenerarcaloryelectricidad.Sinembargo,la ligninatambiénpuedeserutilizadaparaobtenerproductosdemayor valorañadido(Figura15)comofibradecarbono,plásticosyelastómeros termoplásticos,espumasymembranaspoliméricas(Ragauskas,2014;Ten yVermerris,2015). Algunasfraccionesdeligninaseusanparaobtenercombustiblesy productosquímicosqueactualmenteprovienendelpetróleo.Otras fraccionessonutilizadasparaotrasaplicacionesenunagranvariedadde industriascomolamédica,laagrícola,laindustriadeloscombustiblesyla papelera(Figura15). ϭϵ
IIntroducción -Pretratamientosalcalinos:losmáscomunessonlosqueutilizan hidróxidodesodio,potasio,calciooamonio.Laadicióndeun agenteoxidante(oxígeno/H2O2)paraeltratamientoprevioalcalino (NaOH/Ca(OH)2)puedemejorarelrendimientoalfavorecerla eliminacióndelalignina(Carvalheiroetal.,2008).Encomparación conotrospretramientosquímicos,lahidrólisisalcalinaserealizaa menortemperaturaypresión,provocandounamenordegradación deazúcaresjuntoconlaformacióndecompuestosinhibidores (Carvalheiroetal.,2008),ylostiemposdereacciónsondemuchas horas,inclusosemanas(Balietal.,2015).Sehademostradoquees máseficazenresiduosagrícolasqueenmaderas(Kumaretal., 2009). -Ozonólisis:estepretratamientoutilizaozono,queesunpotente oxidantequemuestraunaaltaeficaciaenladeslignificación.La ozonólisistienecomoventajaquetraslareacciónnoseforman compuestosinhibidoresquepuedanafectaralaposteriorhidrólisis yfermentación.Sinembargo,tienecomoinconvenientela utilizacióndeunagrancantidaddeozonoquehaceinviable económicamenteelproceso(SunyCheng,2002). -Organosolv:consisteenlautilizacióndesolventesorgánicos acuososcomometanol,etanol,acetona,etilenglicolyalcohol tetrahidrofurfurílicoconelobjetivodesolubilizarlaligninay proporcionarcelulosaadecuadaparalahidrólisisenzimática(Zhao etal.,2009).Entreellos,eletanoleselsolventemásfavorable porqueespocotóxicoyfácilderecuperar.Comoventajacabe destacarlarecuperacióndeunaligninarelativamentepuraparasu utilizacióncomosubproducto(Zhaoetal.,2009).Como inconvenienteencontramoselaltopreciodelosdisolventesylos posiblesefectosinhibitoriosdeestossobrelahidrólisisenzimáticay losmicroorganismosfermentativos(SunyCheng,2002). -Líquidosiónicos:loslíquidosiónicossonsalesorgánicasque generalmenteseencuentranenestadolíquidoatemperatura Ϯϲ
Introducción I ambientedebidoasubajopuntodefusión.Características intrínsecastalescomoaltaconductividadiónica,altapotenciade solvatación,estabilidadtérmica,inflamabilidad,bajavolatilidady reciclabilidad,lesconfierenelestatusde"disolventesverdes",dado quenoformangasestóxicosoexplosivos.Estoslíquidosdisuelven simultáneamentelaligninayloshidratosdecarbono,ycomo consecuenciasereducealmínimolaformacióndeproductosde degradación,debidoaquelasinteraccionesnocovalentesentrelos productosprincipales,celulosa,hemicelulosasylignina,se interrumpeneficazmente.Parasuaplicaciónanivelindustriales indispensableeldesarrollodemétodosderecicladodeestos líquidos,juntoconlarecuperacióndehemicelulosasyligninatrasla extracciónpreviadecelulosa(Hayes,2009). (c)PretratamientosfísicoͲquímicos -Explosiónconvapor(steamexplosion):estetratamientoesel empleadomayormenteenlaindustriadeproduccióndebioetanol. Elprocesosellevaacaboinyectandovaporaaltapresión(20Ͳ50 bares)enunrecipientedondeseintroduceelsustratoatratar,a unatemperaturade210Ͳ290°C.Labajadadepresiónprovocaque elvaporpenetreenlaestructuralignocelulósicadelmaterialque despuésdeuntiempodereaccióndeterminadoesexpulsadohacia unciclónapresiónatmosférica.Elresultadofinaldeestetipode pretratamientoeslaalteracióndelempaquetamientomicrofibrilar dentrodelaparedcelularylaroturadelafibra,queprovocanun aumentodelaaccesibilidaddelacelulosaalasenzimashidrolíticas. Lascondicionesóptimasdetemperaturaytiempodereacción varíandependiendodeltipodematerial.Comocaracterísticasmás atractivasdestacaelmenorimpactoambiental,menorinversiónde capital,mayorpotencialdeeficienciaenergética,productos químicosycondicionesdelprocesomenospeligrosasyla recuperacióncompletadeloscarbohidratos(AvellaryGlasser, 1998).Comoinconvenientesencontramosladegradaciónparcialde lashemicelulosasylageneracióndealgunoscompuestostóxicos Ϯϳ
IIntroducción quepudieranafectaralassiguientesetapasdehidrólisisy fermentación(Olivaetal.,2003). -Agualíquidacaliente:aligualquelosanteriores,elobjetivo principalessolubilizarlashemicelulosasparahacerquelacelulosa seamásaccesibleyparaevitarlaformacióndeinhibidores.Se realizaatemperaturasmuyelevadas,cercanasalos200°C,y presioneselevadasparamantenerelaguaenestadolíquido.En general,estepretratamientotienecomoventajaqueelaguaactúa comosolvente(noesnecesariouncatalizador)ymediodereacción. Además,reduceelcosteenlaconstruccióndereactoresyaquela corrosiónqueseproduceesmínima.Encuantoalosefectossobre losmaterialeslignocelulósicoscabedestacarlabajaconcentración dehemicelulosassolubilizadas.Elaltocosteenergéticonecesario paracalentarelaguaylautilizacióndegrandescantidadesdeagua, hacenqueestemétodonoseautilizadoactualmenteaescala industrial(Kimetal.,2016). -Explosióndefibrasconamoníaco(ammoniafiberexpansión,AFEX): elpretratamientoconamoníacoseefectúaconcargasdeamoníaco alrededorde1:1(kgdeamoníaco/kgdebiomasaseca)ya temperaturasentre60Ͳ100°Ccontiemposdeprocesomuy variablesdependiendodelatemperaturayaltapresióndurante periodosdetiempo(Alviraetal.,2010).Larápidaliberacióndela presiónproduceunarupturafísicadelmateriallignocelulósicoyuna reduccióndelacristalinidaddelacelulosa.ElmétodoAFEXelimina pocaligninayhemicelulosasperopuedemejorarlaeficaciadela hidrólisisenzimáticasiendonecesariaunamenorcargadeenzimas parallevarlaacabo.Comoventajacabedestacarquenoseproduce formacióndeinhibidoresyqueesunprocesorespetuosoconel medioambiente.ElprocesoAFEXencondicionesóptimaspuede llegaraconseguirunaconversióndemásdel90%decelulosay hemicelulosasenazúcaresfermentables(Teymourietal.,2005).La principaldesventajaesqueaunqueelamoníacopuedeser Ϯϴ
Introducción I recuperadoyrecicladoenelreactorAFEX,elamoníacolíquidoes muycaroparasuaplicaciónindustrial(Kimetal.,2016). -Oxidaciónhúmeda:estepretratamientosebasaenlautilizaciónde oxígenooairecomocatalizador.Elprocesoconsisteenuna oxidacióndurante10Ͳ15minutosatemperaturascomprendidas entre170y200°Cyapresionesde10a12baresdeO2.En principio,tienecomoventajasunaeliminacióneficazdeligninayla formacióndeunabajacantidaddeinhibidores. -Pretratamientoconmicroondasyultrasonido:enelprimerose realizaunainmersióndelmateriallignocelulósicoenreactivos químicosdiluidos,posteriormenteseexponeaunaradiaciónde microondasatiemposentre5Ͳ20minutos(Keshwani,2009).El segundosebasaenlaaplicacióndelultrasonidosobrelabiomasa Algunosautoresyahandemostradolaefectividaddeeste pretretamientorepresentadaenunamejoradelasacarificaciónde lacelulosa(Yachmenevetal.,2009). -ExplosiónconCO2:esunmétodosimilaralaexplosiónconvapor, peroutilizaCO2comogas.Tienecomoventajaselbajocoste,quela toxicidadesnulaaligualquelainflamabilidaddelgas,fácil recuperaciónyunbajoimpactomedioambiental(Taherzadehy Karimi,2008).LaliberaciónexplosivadeCO2apresiónincrementael áreasuperficialdelosmaterialeslignocelulósicos(Alviraetal., 2010).AdemáselCO2puedeformaracidocarbónicoenpresencia dehumedad,elcualfavorecelahidrólisisdelospolímeros(Suny Cheng,2002). (d)Pretratamientosbiológicos Ademásdelospretratamientosfísicos,químicosyfísicoͲquímicosexisten pretratamientosbiológicosbasadosenelusodehongosligninolíticoso susenzimas.EstosmétodosutilizadosenlapresenteTesissedescriben conmásdetalleenelApartadoI.4. Ϯϵ
IIntroducción I.3.2.2.Hidrólisis Laproduccióndebioetanolapartirdemateriallignocelulósicosebasaen laconversióndeloscarbohidratoscelulosayhemicelulosasenazúcares simples(glucosa,xilosa,arabinosa,galactosa,…)queposteriormenteson fermentadosaetanolporunaseriedemicroorganismos.Lahidrólisis puedeserllevadaacabotantoquímicacomoenzimáticamente(Kammet al.,2007). Paralahidrólisisácidaseempleanlosácidossulfúrico,clorhídrico, fluorhídricoynítrico.Lautilizacióndeestosácidosconcentradosgenera problemas,tantoenlacorrosióndelamaquinaria,comodebidoasu toxicidad(VelmuruganyMuthukumar,2011).Debidoaesto,losprocesos dehidrólisissesuelenllevaracaboconácidosdiluidos,normalmente sulfúricooclorhídrico,atemperaturascercanasa200qC.Elrendimiento enlaobtencióndeazúcaressimpleseslimitadoenlahidrólisisácida debidoaquelosazúcarestambiénseconviertenenproductosde degradación,como5Ͳhidroximetilfurfuralapartirdelaglucosayfurfurala partirdexilosa. Debidoalbajorendimientodelahidrólisisácida,sesueleusarlahidrólisis enzimáticaconcelulasasquecatalizanlahidrólisisdelacelulosaen glucosayes100%selectiva.Estasenzimassonproducidasporvarios microorganismos,peroelmáscomúneselhongoTrichodermareesei, aunquetambiénelhongoAspergillusnigerylabacteriaClostridium cellulovoranslasproducen(Araietal.,2006). Debidoalascaracterísticasestructuralesdelacelulosa,nopodemos obtenerdemaneradirectaglucosaporlaaccióndeunacelulasa.Es necesarialaaccióndeendoͲ yexoͲglucanasasyEͲglucosidasas,enzimas queactúansobrelacelulosademanerasimultáneaysinérgica.Las endoglucanasasrompenfragmentosdecelulosadesdeelinterior, mientrasquelaexoglucanasaslohacendesdelosextremosdelacadena; conlaaccióndeambasenzimasseformanmoléculasdecelobiosaqueson hidrolizadasporlaEͲglucosidasaenmoléculassimplesdeglucosa. ϯϬ
Introducción I I.3.2.3.Fermentación Lafermentaciónesunprocesoqueconviertelosmonosacáridos(hexosas ypentosas)enetanolmedianteelusodemicroorganismos.El microorganismoidealdebeproducirunaltorendimientoenlaproducción deetanol,toleraraltasconcentracionesdeésteydelosinhibidores producidosduranteelprocesoytenerlacapacidaddefermentarazúcares aaltastemperaturas(Araietal.,2006).Losorganismosmásusadassonlas levaduras,comoSaccharomycescerevisiaeyCandidautilis,ybacterias, comoZymomonamobilis;estosmicroorganismosposeenaltatoleranciaal etanolyaltosrendimientosdeproducción,entreun90Ͳ97%. I.4.BIODEGRADACIÓNENZIMÁTICADELALIGNINA Comosedescribióanteriormente,laproduccióndebioetanolapartirde materiallignocelulósicorequiereladeconstruccióndelaparedcelularen polímerosindividuales,ylahidrólisisdeloscarbohidratosenazúcares.La cantidaddelignina,asícomosucomposiciónyestructurasonunodelos principalesfactoresquecausanlarecalcitranciadelabiomasa(Studeret al.,2011). Laligninaesunpolímerotridimensionalconstituidoporsubunidadesde fenilpropanounidasentresíporunavariedaddeenlaceséterycarbonoͲ carbono.Laligninaestáíntimamenteentrelazadaconhemicelulosasenla paredcelulardelaplantaformandounamatrizquecubrelasmicrofibrillas decelulosacristalina.Sunaturalezaaromáticayestructuracomplejahace queladegradacióndelaligninaseamuydifícil.Laligninadificultael accesoalascelulasasytambiénseuneaellasproduciendosuinactivación (Rahikainenetal.,2013).Comosehadescritoanteriormente,el pretratamientodelabiomasaparaeliminaromodificarlaligninaes esencialparamejorarlahidrólisisenzimáticadelospolisacáridos.Además delospretratamientosfísicosoquímicos,tambiénseestándesarrollando pretratamientosbiológicosparadeconstruirlabiomasalignocelulósicay eliminarlalignina(Alviraetal.,2010).Lamayoríadelospretratamientos biológicosempleanhongosquedegradanlaligninayquepertenecenal grupodebasidiomicetosdepodredumbreblanca,perotalestratamientos 3ϭ
IIntroducción previosrequierenlargosperíodosdeaplicaciónyconsumenunafracción delospolisacáridosdelaplanta,reduciendoelrendimiento.Sinembargo, elusodeenzimasligninolíticasprocedentesdeestoshongospuede contribuiraladeconstruccióndebiomasavegetalalproporcionarnuevos biocatalizadorescapacesdedegradaromodificarlalignina. Enladegradaciónnaturaldelaparedcelularvegetalhayvariostiposde enzimasinvolucradas.Esteesunprocesosecuencialenelquela eliminacióndeligninaesamenudoelprimerpasoyelquelimitala velocidad.Unavezqueseeliminalaprotecciónconferidaporlalignina recalcitrante,lospolisacáridosdelaparedcelularsonsusceptiblesal ataquedelasenzimashidrolíticas.Lasúnicasenzimasconocidascapaces dedegradarlaligninasonlasutilizadasporlosbasidiomicetos ligninolíticos,losllamadoshongosdepodredumbreblancadebidoalcolor blanquecinodelsustratoenriquecidoconcelulosadespuésdela eliminacióndelalignina.Estosorganismoshandesarrolladoun mecanismobasadoenoxidorreductasasdealtopotencialredox.Estas enzimasincluyenlacasas(fenoloxidasasfúngicas)yperoxidasas ligninolíticas,comolaligninaperoxidasa(LiP),lamanganesoperoxidasa (MnP)olaperoxidasaversátil(VP),queactúanensinergiaconoxidasas productorasdeH2O2,entreotrasenzimas.Ladegradacióndelaligninapor lasoxidorreductasasanterioressedefiniócomouna"combustión enzimática"(KirkyFarrell,1987)debidoalataqueenzimáticoinespecífico, queestádirigidohaciaelanillobencénicodelasestructurasdelalignina. Debidoalanaturalezavoluminosadelalignina,lasperoxidasasque degradanlaligninahandesarrolladounmecanismodignodemenciónque permitelaoxidacióndelpolímeroenlasuperficiedelaenzimaporun radicalproteicoexpuesto,yposteriortransferenciadeelectronesdelargo alcancealcofactordelhemoactivadoporperóxido(RuizͲ Dueñasy Martínez,2009). Laslacasas(fenoloxidasas,EC1.10.3.2)sonoxidasasmulticobreque oxidanfenolessustituidosutilizandooxígenomolecularcomoelaceptor finaldeelectrones(Figura17).Laaccióndirectadelaslacasassobrela ligninaestá,enprincipio,restringidaaunidadesfenólicasquesólo representanunpequeñoporcentajedelpolímerototal,unhechoque 3Ϯ
Introducción I limita su aplicación biotecnológica (Riva, 2006). Una alternativa es el uso de mediadores redox, compuestos simples que forman radicales difusibles estables, que una vez oxidados por la enzima, actúan a distancia e incluso cuando el acceso al polímero de lignina está limitado por impedimentos estéricos (como la estructura compacta de la pared celular de la planta en materiales no deteriorados). El descubrimiento de algunos compuestos sintéticos que pueden actuar como transportadores de electrones entre la enzima y el sustrato final, como es el caso del 1 hidroxibenzotriazol (HBT), ha ampliado la utilidad de las lacasas. Varios estudios han confirmado el potencial de los sistemasmediadores de lacasa para la deslignificación de pasta de papel (Babot et al., 2011), el control del pitch (Gutiérrez et al., 2009), y otras aplicaciones en la industria forestal (Widsten y Kandelbauer, 2008) así como en la producción de bioetanol a partir de lignocelulosa pretratada física y/o químicamente (Palonen y Viikari, 2004b). Figura 17. Mecanismo de actuación del sistema lacasa mediadorsobre la lignina. La capacidad de lacasas de alto potencial redox aisladas de los basidiomicetos Trametes villosaoPicnoporus cinnabarinus (Figura 18), en presencia de HBT como mediador, para eliminar la lignina tanto de plantas madereras (eucalipto) como de plantas anuales (hierba elefante), haciendo que la celulosa sea más accesible a la hidrólisis enzimática, ha sido recientemente demostrada (Gutiérrez et al., 2012; Rico et al., 2014a,b). Posteriormente, también se demostró la capacidad de una lacasa de bajo potencial redox, aislada del ascomiceto Myceliophthora thermophila, en presencia de mediadores fenólicos, para eliminar/modificar la lignina de madera de eucalipto y mejorar la sacarificación enzimática (Rico et al., 2014a). 3 O2 H2O Lacasaoxi Lacasared Mediadorred Mediadoroxi Ligninaoxi Lignina Productos de degradación
IIntroducción Figura18.Representacióntridimensionaldelaestructuramoleculardelalacasadel hongoPycnoporuscinnabarinusmostrandolasregionesenhéliceĮ(rojo),hoja plegadaE(cian),losgirosE(verde)ylosátomosdecobrecatalíticosencolorazul (Prasadetal.,2011). I.5.INFLUENCIADELALIGNINAENLASACARIFICACIÓNDELMATERIAL LIGNOCELULÓSICO Elcontenidoenligninaesunodelosfactoreslimitantesenla digestibilidadenzimáticadelacelulosa(Ding,Liuetal.2012).Lapresencia deligninaresidualtraslospretratamientosconvencionalesproduceun efectonegativoenlaproduccióndeetanoldebidoalossiguientes mecanismos: ϯϰ
Introducción I -Laligninapuedeunirsealascelulasasempleadasparala sacarificación,demaneranoespecífica(bloqueandolossitios activoseimpidiendolauniónalacelulosa),dandolugarala reduccióndelaactividadcatalíticadeestasenzimas(Mansfield etal.,1999;Erikssonetal.,2002;Moilanenetal.,2011). -Laligninaquepermaneceunidaalacelulosatraslos pretratamientosreduceeláreasuperficialdisponibleparala hidrólisisenzimáticadelacelulosa(Moilanenetal.,2011). -Losproductosderivadosdelaligninacomolosfenoles aromáticosácidosyaldehídos,sonamenudotóxicosparalas levadurasquellevanacabolafermentación.Sehadescubierto quepequeñasconcentracionesdeestosinhibidoresdestruyenla integridaddelsistemademembranadelalevadura,impidiendo elcrecimientodeésta(Palmqvistetal.,1999;PalmqvistyHahnͲ Hägerdal,2000;Himmeletal.,2007). Durantelahidrólisisenzimáticademateriallignocelulósicopretratado,las celulasasseunenalasuperficiedelalignina(Palonenetal.,2004).Esta uniónseconsideraperjudicialtantoenelprocesodehidrólisisdecelulosa porlasenzimas,comoenlarecuperaciónenzimática.Lasinteracciones ligninaͲcelulasaestánsiendoestudiadas,sinembargolarazón fundamentalqueexplicaelefectonegativodelaligninaenlahidrólisis enzimáticaaúnnosesabe.Sehanrealizadoestudiosdelefectoinhibitorio delaligninapretratadaconsteamexplosion(Rahikainenetal.,2013). Envariosestudios(Nakagameetal.,2010;Nakagameetal.,2011),seha observadoquelaligninaaisladadeespeciesmadererasmuestrauna mayorinhibiciónenlahidrólisisenzimáticadecelulosaAvicelquela ligninaextraídadeunaplantaherbácea(ej.maíz).Estosugierequela diferentecomposiciónenlaligninanativatambiénpuedeinfluirenla inhibicióndelascelulasas. EnlapresenteTesisseestudióelsistemalacasaͲmediadorcomo pretratamientoparaladeslignificacióndematerialeslignocelulósicoscon ϯϱ
III. RESULTADOS GENERALES Y DISCUSIÓN Cultivos de caña de azúcar (Saccharum sp .)
ResultadosGeneralesyDiscusiónIII EnlapresenteTesissehaestudiado,porunlado,laefectividaddel sistemalacasaͲmediadorcomopretratamientoenzimáticopara deslignificarymejorarlasacarificacióndediversosmateriales lignocelulósicosconvistasalaobtencióndebioetanoldesegunda generación,yporotroladolasinteraccionesentrelaligninaylascelulasas usadasparalahidrólisisenzimática,queconllevaunareduccióndesu actividadcatalítica. III.1.PRETRATAMIENTOSENZIMÁTICOSCONELSISTEMALACASAͲ MEDIADOR Seestudióelefectodepretratamientosenzimáticosbasadosenelsistema lacasaͲmediadorsobrediferentesmaterialeslignocelulósicosdeinterés industrial,conelfindeeliminary/omodificarlaligninapresenteenlas materiasprimasparaobtenerunamayoraccesibilidaddelascelulasasala celulosaconvistasamejorarlasacarificaciónyelrendimientoen bioetanolcelulósico.Asuvez,seanalizaronendetallelasmodificaciones estructuralesquesufreelpolímerodeligninadurantedichos pretratamientosmedianteResonanciaMagnéticaNuclearbidimensional (2DͲNMR)paraconocerelmecanismodedegradación. Estosestudiosserealizaronsobrediferentesmaterialeslignocelulósicos talescomolapajadetrigoyelbagazoylapajadecañadeazúcar, residuosabundantesdelaactividadagraria,asícomosobremaderade paulownia,unaespeciedecrecimientorápido. III.1.1.Pretratamientoenzimáticodepajadetrigoconlacasade PycnoporuscinnabarinusyHBT Lospretratamientosenzimáticossobrepajadetrigoserealizaronconel sistemalacasaͲmediador,seguidodeunaextracciónalcalina.Paraello,se utilizólalacasadeP.cinnabarinusadiferentesdosis(13y65U·gͲ1)y comomediadorseutilizóHBTendiferentesconcentraciones;5,10y20%. Losresultadosobtenidosdemostraronlaeficaciadelpretratamiento enzimáticoenlamodificaciónyeliminacióndeligninadepajadetrigo.El ϰϱ
IIIResultadosGeneralesyDiscusión mejorresultadoseobtuvocuandoseutilizó65U·gͲ1delacasayHBTal 20%,seguidodeunaextracciónalcalinaposterior,produciéndoseuna reduccióndel45%enelcontenidodeligninarespectoalamateriaprima sinpretratar.Elpretratamientoutilizandoenzimasola(65U·gͲ1)sin mediadorconsiguióunareduccióndelcontenidoenligninadel18%, demostrandolaeficaciadelaenzimaaisladamente,aunqueenmenor medida.Porotrolado,elpretratamientoconelsistemalacasaͲmediador enlascondicionesmencionadasanteriormente,perosinextracción alcalinaposterior,produjoundescensodel37%delcontenidodelignina, indicandolabuenaeficaciadeestesistemaenzimáticoparareducirel contenidodeligninaenpajadetrigo. Estosestudiostambiénevidenciaronquelaeliminacióndeligninaenla pajadetrigoestádirectamenterelacionadaconunincrementoenla obtencióndeglucosatraslahidrólisisenzimática(sacarificación).Parala hidrólisisdelospolisacáridosseusóuncóctelenzimáticodelaempresa Novozymes,quecontieneexoͲyendocelulasas(Celluclast2FPU·gͲ1)yɴͲ glucosidasa(6U·gͲ1).Seobservóunamejoradel60%enlaliberaciónde glucosaenlasacarificacióndelapajadetrigopretratadaconelsistema lacasaͲmediadoradosisaltas(65U·gͲ1lacasayHBTal20%),comose indicalaTabla2. Estudiosanterioresindicaronquelaligninadelapajadetrigoutilizadaen estostratamientossecaracterizaporpresentarunidadespͲhidroxifenilo (H),guayacilo(G)ysiringilo(S),estandoenriquecidaenunidadesG(H:G:S, 6:64:30),conunclaropredominiodeunidadesG.Además,laligninadela pajadetrigotambiénincorporaimportantescantidadesdetricina,lacual actúacomounverdaderomonómero,asícomoácidospͲcumáricoy ferúlico(delRíoetal.,2012).Lasmodificacionesestructuralesproducidas enelpolímerodeligninaduranteelpretratamientoenzimáticose estudiaronendetallemedianteResonanciaMagnéticaNuclear bidimensional(2DͲRMN).EnlaFigura19semuestranlosespectrosde HSQC2DͲNMRdelapajadetrigotratadaconelsistemalacasaͲmediador (65U.gͲ1delacasayHBT20%),ytrassersometidaaunaextracción alcalinaposterior,ylosespectroscorrespondientesalapajadetrigo control. ϰϲ
ResultadosGeneralesyDiscusiónIII Tabla2.Contenidoenlignina(determinadacomoligninaklason)yporcentajedeglucosa liberadatraslahidrólisisenzimáticadelasmuestrasdepajadetrigopretratadasporelsistema lacasaͲmediador(P.CinnabarinusyHBT),asícomodelamateriaprimainicial. MuestrasdepajadetrigoLignina(%)Glucosa(%) Materiaprimainicial16.0±0.321.6±0.2 ͲLacasaͲmediador Control15.6±0.224.1±0.0 Lacasa(65UͼgͲ1)15.0±0.129.0±0.1 Lacasa(65UͼgͲ1);HBT(20%)9.8±0.333.6±0.2 ͲLacasaͲmediador+extracciónalcalina Control14.8±0.228.4±0.4 Lacasa(65UͼgͲ1)12.1±0.331.1±0.3 Lacasa(13UͼgͲ1);HBT(10%)9.4±0.135.1±0.5 Lacasa(65UͼgͲ1);HBT(5%)9.2±0.436.5±0.3 Lacasa(65UͼgͲ1);HBT(20%)7.7±0.241.5±0.7 Lacasa(65UͼgͲ1);HBT(20%)/T207.5±0.245.5±0.6 ElanálisisdelasregionesaromáticasdelosespectrosHSQCrevelóqueel tratamientoconelsistemalacasaͲmediadorproduceunaeliminaciónde lasunidadesH,GySdelignina,ademásdelosácidospͲcumáricoy ferúlico,yunaumentoparalelodelasunidadesdeligninaoxidadas(G’y S’).Cabedestacarlaresistenciadelatricinaalpretratamientoenzimático, aunqueseobservaunligerodescensodeesta.Porotrolado,elanálisisde lasregiónesalifáticasdelosespectrosHSQCmostróundescensoenlas señalescorrespondientesalosenlacesdelignina(ɴͲOͲ4´,ɴͲɴ´yɴͲ5´), indicandoladespolimerizacióndelalignina,mientrasquelasseñalesde carbohidratos(X:xilosayGl:glucosa)nosevieronafectadasporel tratamiento.Enconclusión,larupturadelosenlacesdentrodelpolímero deligninayelaumentodelasunidadesdeligninaoxidadaponende manifiestoqueelsistemalacasaͲmediadorsigueunmecanismode despolimerizaciónoxidativa. ϰϳ
III ResultadosGeneralesyDiscusión Figura19.Regiónesalifáticas(ayc)yaromáticas(byd)delosespectrosHSQC2DͲNMRdelas muestrasdepajadetrigotratadaconelsistemalacasaͲmediador(cyd),utilizando65UͼgͲ1de lacasayHBT20%,ydesucorrespondientecontrol(sinenzimanimediador).Principales estructurasidentificadasenlosespectrosHSQC:EͲOͲ4഻alquilariléter(A),EͲ5഻fenilcumarano (B),EͲE഻resinol(C),tricina(T),ácidopͲcumárico(PCA),ácidoferúlico(FA),alcoholcinamílico terminal,unidadespͲhidroxifenilo(H),unidadesguayacilo(G),unidadessiringilo(S),unidades Goxidadas(G഻)yunidadesSoxidadas(S഻). III.1.2.Pretratamientoenzimáticodebagazoypajadecañadeazúcar conlacasadePycnoporuscinnabarinusyHBT ElmismosistemalacasaͲmediadorutilizadoconlapajadetrigofue evaluadosobreelbagazoylapajadelacañadeazúcar,cuyasligninas presentanunacomposiciónyestructuramuydiferentes.Mientrasquela ligninadelbagazodecañadeazúcarcontienepredominantemente ϰϴ
ResultadosGeneralesyDiscusiónIII unidadesdetipoSyenlacesɴͲéternocondensados,laligninadelapaja estáenriquecidaenunidadesdetipoGypresentaenlacesmás condensados(delRíoetal.,2015).Esteestudionospermitióconocerel efectodelacomposicióndelaligninasobrelaeficienciadel pretratamientoconelsistemalacasaͲmediador. ElpretratamientoconlacasadeP.cinnabarinusyHBTsobrelosresiduos delacañadeazúcarnofuetanefectivoenlaeliminacióndeligninacomo enelcasodelapajadetrigo.Porello,alternativamente,seevaluóun pretratamientoqueconsistióenusarmenosdosisdeenzimaymediador, perorepitiendoeltratamientoencuatrociclos.Seutilizaron50U·gͲ1de lacasayHBTal3%encadaunodelos4ciclos.Bajoestascondicionesse consiguióundescensodel27%y31%enelcontenidodeligninaenel bagazoylapajadecañadeazúcar,respectivamente(Tabla3). Tabla3.Contenidoenligninayporcentajedeglucosaliberadatraslahidrólisisenzimáticade lasmuestrasdepajaybagazodecañaazúcartratadasconelsistemalacasaͲmediador(P. cinnabarinusyHBT),asícomodelasmateriasprimasiniciales. MuestrasLignina(%)Glucosa(%) Materiaprimabagazocañadeazúcar ͲLMS+extracciónalcalina(4ciclos) 17.8±0.635.9±0.7 Control17.5±0.440.1±0.2 Lacasa(50UͼgͲ1)16.8±0.344.2±0.4 Lacasa(50UͼgͲ1);HBT(3%)12.8±0.355.8±0.4 Materiaprimapajacañadeazúcar ͲLMS+extracciónalcalina(4ciclos) 17.0±0.234.9±0.2 Control16.6±0.239.2±0.2 Lacasa(50UͼgͲ1)15.1±0.142.3±0.1 Lacasa(50UͼgͲ1);HBT(3%)11.5±0.357.1±0.3 Tambiénseobservóqueeltratamientoconlacasasola,enausenciade HBT,resultómuchomenosefectivo,observándosetansoloundescenso del4%y9%enelcontenidoenlignina,enbagazoypaja,respectivamente. Aligualqueocurriótraselpretratamientodelapajadetrigo,eldescenso ϰϵ
III ResultadosGeneralesyDiscusión enelcontenidodeligninadelosresiduosdelacañadeazúcarllevó tambiénasociadounincrementoenlaobtencióndeglucosatrasla hidrólisisenzimática.Enestecaso,seconsiguióunamejoradel39%y46% enelrendimientodeglucosaparaelbagazoypajadecañadeazúcarpreͲ tratadasconelsistemalacasaͲmediador,encomparaciónconelcontrol. Porotrolado,seestudióendetallelasmodificacionesestructuralesdela ligninadurantelostratamientosenzimáticosmediante2DͲNMR(Figura 20).Tantoenelbagazocomoenlapajadelacañadeazúcar,seobservó undescensosignificativoenlosenlacesycantidaddelignina, confirmándoseelmecanismodedespolimerizaciónoxidativadelalignina. Figura20.RegionesaromáticasdelosespectrosHSQCdelapaja(arriba)yelbagazo(abajo)de lacañadeazúcar,juntoconlasprincipalesestructurasdeligninaidentificadasenlosmismos. Losespectrosa)yc)correspondenaloscontrolesmientrasquelosespectrosb)yd) correspondenalasmuestrastratadas. ϱϬ
ResultadosGeneralesyDiscusiónIII III.1.3.PretratamientoenzimáticodePaulowniafortuneiconlacasade Myceliophthorathermophilaysiringatodemetilo EltercerpretratamientoconelsistemalacasaͲmediadorestudiadoenla presenteTesisutilizócomosustratolamaderadepaulownia(P.fortunei), unaespeciedecrecimientorápidocuyaligninaestácompuesta principalmenteporunidadesdetipoG(60%)yS(40%)(Rencoretetal., 2009). Enesteestudio,elpretratamientoconsistióen4ciclos,dondecadauno delosciclosincluíaeltratamientoconelsistemalacasaͲmediadorseguido deunaextracciónalcalinaconperóxidodehidrógeno.Adiferenciadelos dosanteriores,elsistemalacasamediadorenestecasoestabacompuesto porlalacasacomercialdelhongoM.thermophila(MtL)ysiringatode metilo(MeS)comomediador.Encadaunodelosciclosseutilizaron50 U·gͲ1deMtLyMeSal3%.Bajoestascondiciones,seconsiguióun descensodetansoloun24%delcontenidodeligninadepaulownia,una reducciónmuchomenorquelaqueseconsiguiócuandosetratómadera deeucalipto(50%dereducción),utilizándoselasmismascondiciones (Ricoetal.,2014).Estadiferenciapuedeserdebidaaladiferente composicióndelaligninaenambossustratos,yaqueenlamaderade eucaliptopredominanlasunidadesdetipoS(relaciónS/Gde3.6)ylos enlacesɴͲéternocondensados(másfácilesdedegradar),mientrasquela ligninadepaulowniacontienepredominantementeunidadesdetipoG (relaciónS/Gde0.8)ypresentaunaestructuraconmásenlacesCͲCy,por tanto,máscondensada. Aligualqueocurrióconotrasmateriaprimaspretratadas,eldescensoen elcontenidodeligninaestuvoacompañadodeunincrementoenla obtencióndeglucosatraslahidrólisisenzimática.Utilizandoelmismo cóctelenzimáticoqueenloscasosanteriores,seobtuvounamejoradel 40%enlaliberacióndeglucosa(Tabla4). ϱϭ
III ResultadosGeneralesyDiscusión Figura26.SensogramadeSPRqueestudialaadsorcióndeCtec2(5mg/mL)disueltoen solucionesadiferentesconcentraciones50mMy200mM,sobreambasMWLs.Laenzimase introdujoenelminuto1yenelminuto5seproduceellavadoconsolucióntampónalas correspondientesconcentraciones. Enconclusión,losdiversosestudiosrealizadosenlapresenteTesishan demostradoelgranpotencialdelsistemalacasaͲmediadorcomo pretratamientoparaladeslignificacióndediferentesmateriales lignocelulósicosconlaconsiguientemejoraenlaposteriorsacarificación enzimáticaconvistasamejorarelrendimientoenbioetanol.Porotro lado,tambiénsehandemostradolosefectosnegativosdelaligninaenla inhibicióndelascelulasas. ϱϴ
IV. REFERENCIAS Cultivos de eucalipto ( Eucalyptus globulus)
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V. PUBLICACIONES Cultivos de Picea abies
Publicaciones V PUBLICACIÓN 1: Rencoret J., Pereira A., del Río J.C., Martínez A.T., Gutiérrez A. (2016) Laccase-mediator pretreatment of wheat straw degrades lignin and improves saccharification. BioEnergy Research 9: 917-93. 77
Laccase-Mediator Pretreatment of Wheat Straw Degrades Lignin and Improves Saccharification Jorge Rencoret 1 &Antonio Pereira 1 &José C. del Río 1 &Angel T. Martínez 2 & Ana Gutiérrez 1 Published online: 12 May 2016 #Springer Science+Business Media New York 2016 Abstract Agricultural by-products such as wheat straw are attractive feedstocks for the production of second-generation bioethanol due to their high abundance. However, the presence of lignin in these lignocellulosic materials hinders the enzymatic hydrolysis of cellulose. The purposes of this work are to study the ability of a laccase-mediator system to remove lignin improving saccharification, as a pretreatment of wheat straw, and to analyze the chemical modifications produced in the remaining lignin moiety. Up to 48 % lignin removal from ground wheat straw was attained by pretreatment with Pycnoporus cinnabarinus laccase and 1- hydroxybenzotriazole (HBT) as mediator, followed by alkaline peroxide extraction. The lignin removal directly correlated with increases (∼60 %) in glucose yields after enzymatic saccharification. The pretreatment using laccase alone (without mediator) removed up to 18 % of lignin from wheat straw. Substantial lignin removal (37 %) was also produced when the enzyme-mediator pretreatment was not combined with the alkaline peroxide extraction. Two-dimensional nuclear magnetic resonance (2D NMR) analysis of the whole pretreated wheat straw material swollen in dimethylsulfoxide-d 6 revealed modifications of the lignin polymer, including the lower number of aliphatic side chains involved in main β-O-4′ and β-5′inter-unit linkages per aromatic lignin unit. Simultaneously, the removal of p-hydroxyphenyl, guaiacyl, and syringyl lignin units and of p-coumaric and ferulic acids, as well as a moderate decrease of tricin units, was observed without a substantial change in the wood polysaccharide signals. Especially noteworthy was the formation of Cα-oxidized lignin units during the enzymatic treatment. Keywords Enzymatic delignification .Laccase-mediator . Lignin .Wheat straw .2D NMR .Bioethanol Introduction Agricultural and forestry residues represent an enormous source of readily available biomass for biofuel production without the need for additional land cultivation. Among agricultural residues, wheat straw is potentially one of the most favorable feedstocks in terms of the quantity of biomass available [1]. However, in spite of the availability of these residues or other lignocellulosic biomass, their variable composition and recalcitrance represent some technical and economic challenges. Cellulose, hemicelluloses, and lignin are the three main components of lignocellulosic biomass linked into a complex matrix highly resistant to chemical and biological conversion. These components are more resistant to being broken down and fermented than starch and sucrose in the conventional food crops, making the conversion processes more complicated. Biofuel production from lignocellulosic material requires deconstruction of the cell wall into individual polymers and hydrolysis of the carbohydrates into monomeric sugars. One of the major factors causing biomass recalcitrance toward saccharification is correlated with the content and composition of lignin [2–4]. Lignin is a three-dimensional polymer constituted by phenylpropanoid subunits linked together by different ether and carbon-carbon bonds. Lignin is intimately interlaced with Jorge Rencoret and Antonio Pereira contributed equally to this work. *Ana Gutiérrez [email protected] 1 Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, Reina Mercedes 10, 41012 Seville, Spain 2 Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain Bioenerg. Res. (2016) 9:917–930 DOI 10.1007/s12155-016-9745-z ϳϵ
hemicelluloses in the plant cell wall forming a matrix to cover the largely crystalline cellulose microfibrils. Its aromatic nature and complex structure make lignin degradation very difficult. Lignin has been shown to have a detrimental effect on the hydrolysis of biomass because it physically hinders the access of cellulases and also binds them reducing activity [5]. Therefore, biomass pretreatment to remove lignin is essential for the enzymatic hydrolysis of lignocellulose. Biotechnology can contribute to plant biomass deconstruction by providing biocatalysts being able to degrade or modify lignin [6]. Due to the complex structure of lignin, including heterogeneity of the monomers and linkages, which are not hydrolyzable, these biocatalysts must be oxidative (i.e., oxidoreductases) and also non-specific. Among these, laccases seem to be the most suitable enzymes for industrial application, because they only require dioxygen as oxidant and also because it can be produced on a large scale [7]. Laccases (phenoloxidases, EC 1.10.3.2) are multicopper oxidases that oxidize substituted phenols using molecular oxygen as the final electron acceptor. The direct action of laccases on lignin is, in principle, restricted to the phenolic units that only represent a small percentage of the total polymer [8], a fact that limits their biotechnological application. However, the discovery that some synthetic compounds can act as electron carriers between the enzyme and the final substrate [9], 1-hydroxybenzotriazole (HBT) being among the most efficient ones [10], has expanded the utility of laccases. A number of studies have confirmed the potential of laccasemediator systems for paper pulp delignification [11,12], pitch control [13], polymer modification [14], and other applications in the forest industry [15] and bioethanol production from pretreated lignocellulose [16–18]. Recently, the ability of fungal laccases to remove lignin (when applied in combination with redox mediators) from whole [19,20] and ensiled [21] lignocellulosic biomass, making cellulose accessible to hydrolysis, was reported. Additionally, the use of laccases in bioethanol production has also been reported as a biotechnological tool for the removal of phenolic inhibitors generated during steam explosion of lignocellulosic feedstocks [22], although some recent work also shows that some laccasederived compounds can affect negatively the enzymatic hydrolysis [23]. A previous work on the laccase-mediator treatment of acidpretreated wheat straw has been reported [17]. The novelty of the pretreatment described here, based on the use of a fungal laccase from the basidiomycete Pycnoporus cinnabarinus [24] in combination with HBT as mediator [25], is that it was applied directly in the ground wheat straw feedstock (without a previous chemical pretreatment). Additionally, in the present study, the modification of lignin structure in the pretreated lignocellulosic material was analyzed in depth by two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy of the whole sample at the gel state [26,27]. Besides lignin modification and removal, the effect of the laccase-mediator on the saccharification yield from the pretreated wheat straw feedstock was also assessed. Material and Methods Lignocellulosic Feedstock, Enzyme, and Mediator Wheat straw (Triticum durum var. Carioca) was harvested from an experimental field in Seville (Spain). Wheat straw samples were air-dried and grounded in an IKA MF10 cutting mill to pass through a 100-mesh screen and then finely milled using a Retsch PM100 planetary mill at 400 rev min −1 (with 5- min breaks after every 5 min of milling) using a 500-mL agate jar and agate ball bearings (20× 20 mm). The total ball-milling time for the samples was 5 h. The chemical composition of wheat straw feedstock (as % dry weight) was as follows: glucose, 39.4± 0.7; xylose, 16.0 ± 0.3; arabinose, 3.8 ± 0.2; soluble acid lignin, 1.5 ± 0.1; and Klason lignin, 16.0± 0.3. The laccase was provided by INRA (Marseille, France) and was obtained from a laccase-hyperproducing strain of the fungus P. cinnabarinus (Herpoël et al. 2000). Its activity was measured as initial velocity during oxidation of 5 mM ABTS from Roche to its cation radical (ε 436 29300 M −1 cm −1 ) in 0.1 M sodium acetate (pH 5) at 24 °C. The laccase activity of the enzyme preparation was 102 U/ml (specific activity 156 U/mg). One activity unit (U) was defined as the amount of enzyme transforming 1 μmol of ABTS per minute. HBT from Sigma-Aldrich (Steinheim, Germany) was used as mediator. Laccase-Mediator Treatments The wheat straw samples were treated with the P. cinnabarinus laccase in the presence (and absence) of HBT, as mediator. Laccase doses of 13 and 65 U g −1 were assayed, together with 5, 10, and 20 % HBT, all referred to straw dry weight. The treatments were carried out in 200-mL pressurized bioreactors (Labomat, Mathis) placed in a thermostatic shaker at 170 rev min −1 and 50 °C, using 4-g (dry weight) samples at 6 % (w/w) solid loading in 50 mM sodium tartrate buffer (pH 4) under O 2 atmosphere (2 bar) for 24 h. Additionally, the treatment with laccase (65 U g −1 ) and HBT (20 %) was also performed in the presence of 0.05 % Tween 20, to test the effect of adding a surfactant in both the enzymatic delignification and enzymatic hydrolysis of wheat straw [28]. After the treatment, the samples were filtered through a Büchner funnel and washed with 1 L of water. In some cases, a subsequent alkaline peroxide extraction step was performed after the enzymatic pretreatment. In this case, enzymatically treated samples at 6 % (w/w)solid loading were submitted to a peroxide-reinforced 918 Bioenerg. Res. (2016) 9:917–930 ϴϬ
alkaline extraction using 1 % (w/w)NaOHand3%(w/w) H 2 O 2 (also with respect to sample dry weight) at 80 °C for 90 min, followed by water washing [29]. The solid loading for the latter step was achieved by determining the moisture content of an aliquot. Treatments with laccase (65 U g −1 ) alone (without mediator) and controls without laccase and mediator were also performed (followed in both cases by the corresponding alkaline peroxide extraction). A control with mediator alone was not included taking into account the results from previous studies. Duplicate experiments of a representative (65 U g −1 laccase and 20 % HBT) laccase-mediator treatment (including control, laccase alone and laccase-HBT) were performed to estimate the variability in biological replicates (as shown in Table 1footnote). A one-way analysis of variance (ANOVA) was conducted to compare the effects of the different enzymatic treatments on the lignin removal and on the releases of glucose and xylose. Post hoc pairwise comparisons, using the Tukey HSD test, were performed in order to determine which means are significantly different from each other. Klason lignin content was estimated (in triplicate measurements) according to T222 om-88 [30]. The data from both biological and technical replicates were averaged. Weight loss (%) was determined for all the treatments (Table 1) with respect to the control without enzyme-mediator and alkaline extraction: laccase (65 U g −1 ), 2.3 %; laccase (65 U g −1 )-HBT (20 %), 6.1 %; control/alkaline peroxide, 0.8 %; laccase (65 U g −1 )/alkaline extraction, 4.8 %; laccase (13 U g −1 )-HBT (10 %)/alkaline extraction, 7.3 %; laccase (65 U g −1 )-HBT (5 %)/alkaline extraction, 7.7 %; laccase (65 U g −1 )-HBT (20 %)/alkaline extraction,13.4 %; and laccase (65 U g −1 )- HBT (20 %)/T20/alkaline extraction, 13.5 %. The weight loss of control without enzyme-mediator and alkaline extraction with respect to initial wheat straw was 12.7 %. Saccharification of Treated Wood The laccase-pretreated samples were hydrolyzed with a cocktail containing commercial enzymes (from Novozymes, Bagsvaerd) with cellulase (Celluclast 1.5 L; 2 filter paper units (FPU) g −1 ) and β-glucosidase (Novozyme 188; 6 U g −1 ) activities, at 1 % solid loading in 3 mL of 100 mM sodium citrate (pH 5) for 72 h at 45 °C, in a thermostatic shaker at 170 rev min −1 (in triplicate experiments). The specific activities of Celluclast 1.5 L and βglucosidase are 700 EGU/g and 250 CBU/g, respectively. The different monosaccharides released were determined as alditol acetates [31] by GC. An HP 5890 gas chromatograph (Hewlett-Packard, Hoofddorp, The Netherlands) equipped with a split-splitless injector and a flame ionization detector was used. The injector and detector temperatures were set at 225 and 250 °C, respectively. Samples were injected in the split mode (split ratio 10:1). Helium was used as the carrier gas. The capillary column used was a DB-225 (30 m × 0.25 mm i.d., Table 1 Lignin content and monosaccharide release (% sample weight) by cellulase hydrolysis of wheat straw samples Wheat straw samples Lignin Glucose Xylose Initial wheat straw 16.0 ± 0.3 21.6 ± 0.2 6.9 ± 0.1 - Laccase-mediator: Control 15.6 a ± 0.2 24.1 a ± 0.0 7.3 a ± 0.4 Laccase (65 U g −1 ) 15.0 a ± 0.1 29.0 b ± 0.1 9.4 b ± 0.1 Laccase (65 U ·g −1 )-HBT (20 %) 9.8 b ± 0.3 33.6 c ± 0.2 11.2 c ± 0.2 - Laccase-mediator/alkaline peroxide: Control 14.8 a ± 0.2 28.4 a ± 0.4 9.8 a ± 0.3 Laccase (65 U g −1 ) 12.1 b ± 0.3 31.1 b ± 0.3 9.8 a ± 0.2 Laccase (13 U g −1 )-HBT (10 %) 9.4 c ± 0.1 35.1 c ± 0.5 11.4 b ± 0.6 Laccase (65 U g −1 )-HBT (5 %) 9.2 c ± 0.4 36.5 d ± 0.3 11.5 b ± 0.6 Laccase (65 U g −1 )-HBT (20 %) 7.7 d ± 0.2 41.5 e ± 0.7 13.3 c ± 0.2 Laccase (65 U g −1 )-HBT (20 %)/T20 7.5 d ± 0.2 45.5 f ± 0.6 13.7 c ± 0.0 Lignin content (as Klason lignin, corrected for ash) and monosaccharides from cellulase hydrolysis of wheat straw samples treated with the following: (i) P. cinnabarinus laccase (13 and 65 U g −1 ) and HBT mediator (10 and 20 %); (ii) the same previous enzymatic treatment (i) followed by an alkaline peroxide extraction including a treatment in presence of Tween 20 (T20) and (ii) compared with a control without enzyme, a treatment with laccase alone, and the initial wheat straw sample. Biological duplicates for a representative laccase-mediator treatment (including 65 U g −1 laccase and 20 % HBT) were separately analyzed showing that the 95 % confidence intervals of the biological duplicates are smaller than the differences found between the control, laccase alone, and laccase-HBT treatments (data not shown). In the table, means ± SD from technical triplicates of experiments representative for the different treatments are provided. Letters next to the means, from Tukey test, show whether or not the results from different treatments (shown in each column) are significantly different from each other, at the 0.05 level (means with the same letters are not significantly different, whereas means followed by different letters are significantly different) Bioenerg. Res. (2016) 9:917–930 919 ϴϭ
0.15-μm film thickness; Agilent J&W). The oven was temperature-programmed from 220 °C (held for 5 min) to 230 °C (held for 5 min) at 2 °C min −1 . Peaks were quantified by area, and glucose and xylose were used as standards to elaborate calibration curves. The data from both biological and technical replicates were averaged. 2D NMR Spectroscopy For gel-state NMR experiments, ∼70 mg of finely divided (ballmilled) wheat straw samples was directly transferred into 5-mm NMR tubes and swelled in 1 mL of deuterated dimethylsulfoxide (DMSO-d 6 ), forming a gel inside the NMR tube [26,27]. Heteronuclear single-quantum correlation (HSQC) 2D-NMR spectra were acquired at 25 °C on a Bruker AVANCE III 500- MHz spectrometer fitted with a cryogenically cooled 5-mm TCI gradient probe with inverse geometry (proton coils closest to the sample). The 2D 13 C- 1 H correlation spectra were carried out using an adiabatic HSQC pulse program (Bruker standard pulse sequence “hsqcetgpsisp2.2”), which enabled a semiquantitative analysis of the different 13 C- 1 H correlation signals. Spectra were acquiredfrom10to0ppm(5000Hz)inF2( 1 H) using 1000 data points for an acquisition time of 100 ms, an interscan delay of 1 s, and from 200 to 0 ppm (25,168) in F1 ( 13 C) using 256 increments of 32 scans, for a total acquisition time of 2 h 34 min. The 1 J CH used was 145 Hz. Processing used typical matched Gaussian apodization in 1 H and a squared cosine bell in 13 C. The central solvent peak was used as an internal reference (δ C /δ H 39.5/2.49). The 13 C- 1 H correlation signals from the aromatic region of the spectrum were used to estimate the content of lignin, p-coumaric acid, ferulic acid, and tricin (compared with the amorphous polysaccharide content, estimated from the anomeric xylose and glucose signals) and the lignin composition in terms of G, S, and oxidized S (S') units. Correlations in the aliphatic-oxygenated region were used to estimate the inter-unit linkage and end-unit abundances in lignin. The intensity corrections introduced by the adiabatic pulse program permit to refer the side-chain integrals to the previously obtained number of lignin units. Results and Discussion Delignification of Wheat Straw by Laccase-HBT Wheat straw lignin is a guaiacyl-rich lignin [32]thatisusually reported to be more resistant to degradation than the syringyl type [33]. For this reason, and also based on results from previous studies, a high-redox potential laccase (from the basidiomycete P. cinnabarinus) and mediator (HBT) were selected for wheat straw delignification. Enzymatic pretreatments using different doses of P. cinnabarinus laccase (13 and 65 U g −1 ) and HBT (5, 10, and 20 %) were carried out. The selection of these doses was based on previous studies on the pretreatment of another nonwoody feedstock (elephant grass) [19] with a lignin having also a high proportion of guaiacyl units although not as high as that of wheat straw [32,34]. Additionally, the effect of the combination of the enzymatic treatment with a subsequent alkaline peroxide extraction was studied. The lignin content (as Klason lignin) of wheat straw samples after the pretreatments was determined (Table 1). The effect of oxygen (used in the enzymatic reactions) and alkaline peroxide extraction in wheat straw delignification can be observed by comparing the corresponding controls with the initial straw. The lignin content of wheat straw samples after the laccasemediator pretreatment using P. cinnabarinus laccase (65 U g −1 ) in the presence of HBT (20 %) decreased about 37 % with respect to the corresponding control (treated under the same conditions except the addition of enzyme and mediator) (Table 1). The pretreatment with laccase alone (in the absence of HBT) only decreased the lignin content by 5 %. Similar values of lignin content decrease have been reported after laccase (alone) treatment of several lignocellulosic feedstocks, as steam-pretreated giant reed and Northern spruce [35]. The laccase alone is not very efficient for delignification because of its low oxidation potential and for stearic hindrance reasons. The relatively low oxidation potential of laccases only allows them to oxidize phenolic groups in lignin. Combination of the enzymatic pretreatment with a subsequent alkaline peroxide extraction step (removing partially degraded lignin) increased wheat straw delignification up to 48 and 18 % (with respect to lignin content of the control sample) in the pretreatments with laccase-HBT and laccase alone, respectively (Table 1). With the aim of exploring if the enzymatic delignification could be enhanced by the presence of a surfactant, as reported for pulp biobleaching [36], addition of Tween 20 was tested, but only a modest effect on the delignification of wheat straw was observed. When the enzymatic pretreatment (followed by alkaline peroxide extraction) was carried out under the same reaction conditions and dose of laccase, but with lower dose of HBT (5 %), 38 % reduction of lignin content (with respect to the control sample) was attained, and a similar value (37 % delignification) was obtained when both the laccase and mediator doses were reduced to 13 U g −1 enzyme and 10 % HBT, respectively. Similar delignification degrees to those above described (with lower laccase-mediator doses) were obtained after four sequential laccase-HBT treatments (including four alkaline peroxide extraction steps) of elephant grass [19]. Interestingly, when the latter pretreatment was applied to eucalypt wood, higher delignification degrees (up to 48 %) were attained [19,37,38], showing that some woody feedstocks can be competitive for bioethanol production. The better delignification values obtained in the latter studies can be 920 Bioenerg. Res. (2016) 9:917–930 ϴϮ
related with the predominance of syringyl lignin units in eucalypt wood with respect to elephant grass and wheat straw [32,34,39,40]. On the other hand, no decrease in lignin content was obtained by other authors after treating steamexploded wheat straw with P. cinnabarinus laccase and HBT [41]. Enzymatic Hydrolysis of Pretreated Wheat Straw The wheat straw samples treated with laccase alone and in the presence of HBT and the corresponding controls (and the initial untreated wheat straw) were hydrolyzed (72 h) using a cellulase and β-glucosidase cocktail [19], and the main monosaccharides released (glucose and xylose) were analyzed by GC. At low cellulase (2 FPU g −1 )andβ-glucosidase (6 U g −1 ) doses, increases in glucose and xylose yields up to 40 and 47 % (with respect to controls), respectively, were attained in the samples pretreated with 65 U g −1 of laccase and 20 % mediator (Table 1). Interestingly, in the samples pretreated with laccase alone, 24–25 % increases in glucose and xylose release were produced, although the Klason lignin content was not significantly lowered. The positive effect of laccase (alone) in enzymatic saccharification has been attributed to the binding of laccase to lignin in lignocellulosic substrates that competes with and consequently reduces the non-specific binding of cellulases to lignin improving saccharification [17], in addition to some delignification effect on the phenolic lignin units. Moreover, a surface analysis of lignocellulose substrate after treatment with Trametes laccase revealed an increase in carboxylic acid residues after laccase treatment and suggested that this enzymatic modification to lignin may decrease the non-specific adsorption of negatively charged cellulases [16]. On the other hand, increases up to 46 and 9 % in glucose and 35 and 0 % in xylose yields, respectively, were produced when the enzymatic pretreatments of laccase-HBTand laccase alonewerecombinedwithanalkalineperoxideextractionstep (the highest increases being attained with the highest doses of laccase, 65 U g −1 , and HBT, 20 %). Moreover, a similar improvement in saccharification yield was produced after the laccase-mediator treatment alone (without a subsequent alkaline extraction) unlike in a recent study of a laccase-HBT treatment of acid-pretreated wheat straw [17]. In the pretreatment with 13 U g −1 of laccase, in combination with 10 % mediator (followed by alkaline extraction), the increases in glucose and xylose yields were 24 and 16 % (with respect to control), respectively (Table 1). Similarly, an increase in glucose and xylose yields up to 29 and 17 %, respectively, was produced using 65 U g −1 of laccase, in combination with only 5 % mediator (followed by alkaline extraction). The above glucose release values are higher than those recently reported by other authors in the treatment with laccase-HBT of acidpretreated wheat straw [17] in which higher laccase doses were used but also higher solid loading in enzymatic hydrolysis. Other authors have reported no improvement in saccharification yields after different laccase-mediator treatments of steam-exploded wheat straw [41]. On the other hand, the saccharification yields shown here after laccase-mediator pretreatment of wheat straw are higher than those reported for other pretreatments of wheat straw like steam explosion [42]. The sugar degradation and generation of inhibitory compounds during steam explosion that affect the hydrolysis can explain the lower saccharification yields attained in steamexploded wheat straw. Additionally, it should be mentioned the higher solid loading used by these authors that also may affect enzymatic saccharification. Finally, noteworthy was the improvement in glucose and xylose yields (up to 60 and 40 %, respectively) obtained in the present study when a surfactant (Tween 20) was added during the laccase-HBT pretreatment (Table 1). Since the addition of Tween 20 did not affect the delignification of wheat straw (as shown above) and, in contrast, enhanced the saccharification, its effect may be related with an increase of cellulase action on cellulose [28]. Enzymatic Modification of Wheat Straw Lignin (as Shown by 2D NMR) The modification of lignin structure produced by the enzymatic pretreatments of wheat straw was studied by 2D NMR. With this purpose, the whole wheat straw samples were swelled in DMSO-d 6 forming a gel and analyzed by HSQC NMR. The main lignin structures identified are shown in Fig. 1, and the different lignin signals assigned on the spectra are listed in Table 2. The HSQC spectra of the different straw samples are provided in Figs. 2,3,and4. The composition of lignin, in terms of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units, and the p-coumaric acid and ferulic acid contents, together with the relative abundance of the main inter-unit linkages in the different samples, estimated from the NMR signal volume integrals, are shown in Tables 3and 4. Effect of the Laccase-Mediator Treatment Figure 2shows the HSQC spectra of the wheat straw samples after pretreatment with laccase (65 U g −1 ) in the presence (Fig. 2c, f) and absence (Fig. 2b, e) of HBT (20 %) and the corresponding control (Fig. 2a, d). The aliphatic-oxygenated region of the HSQC spectrum of the control without enzyme (Fig. 2a) shows signals of lignin and carbohydrates, the latter mainly corresponding to amorphous xylan (X), acetylated xylan (X'), and glucan (Gl) units, since crystalline cellulose is nearly “silent”in lignocellulose gel spectra under solution NMR conditions. In this region, signals of methoxyls and side chains in β-O-4′alkyl-aryl ether lignin substructures (A), Bioenerg. Res. (2016) 9:917–930 921 ϴϯ
effect was also produced by the alkaline peroxide extraction itself. If we compare the NMR spectra of controls with (Fig. 3a, d) and without (Fig. 2a, d) alkaline peroxide extraction, it is evident that B and C signals decreased with the alkaline peroxide extraction. Likewise, the intensities of signals of side chains in β-O-4′lignin, especially those where the second unit is a G unit, also decreased. In the aromatic region of the spectrum (Fig. 3d),themostnoticeableeffectofalkaline peroxide extraction was the decrease in FA. Interestingly, the hemicellulose was not deacetylated (unchanged X' 1 ,X' 2 ,and X' 3 signals) in agreement to the mild conditions of the alkaline extraction used. Finally, interesting results were obtained when lower laccase and HBT doses were assayed, always followed by an alkaline peroxide extraction, including the following: (i) reduced mediator dose (to only 25 % of the initial) (Fig. 4b, d) and (ii) reduced both laccase (to 20 % of the initial) and mediator dose (to 50 % of the initial) (Fig. 4a, c). These two treatments seem to be very efficient in degrading/removing lignin (including β-O-4′side chains and lignin units) similarly to the treatment with higher mediator doses, although the latter showed higher efficacy toward H lignin units, PCA, and tricin. In the three treatments, a preferential degradation of G units with respect to S ones was observed (Table 4). The increase in S/G ratio with the treatment was also revealed in previous studies dealing with laccase-mediator pretreatments of other lignocellulosic feedstocks [19,37,38]. On the other hand, in spite of similar lignin removal with both lower doses (Table 1), a higher decrease in S and G lignin units is shown by 2D NMR for the former one that is also correlated with a higher saccharification yield. Conclusions Wheat straw can be delignified by a basidiomycete laccase in the presence of HBT, directly on the ground lignocellulosic material (i.e., without a previous chemical pretreatment) Table 4 NMR analysis after laccase-HBT (different doses) treatment followed by alkaline extraction (parentheses refer to total lignin) Control Laccase Lac13- HBT10 Lac65- HBT5 Lac65- HBT20 - Sample composition a : Syringyl lignin units (S) 4.8 (28) 3.3 (30) 1.3 (24) 0.2 (8) 0.3 (10) C α -oxidized S units (S') 0 0.3 (3) 1.0 (19) 0.6 (29) 1.0 (38) Guaiacyl lignin units (G) 11.6 (68) 6.5 (58) 1.5 (28) 0.7 (37) 0.2 (6) C α -oxidized G units (G') 0 0.3 (3) 1.1 (20) 0 1.1 (39) p-Hydroxyphenyl units (H) 0.8 (5) 0.8 (7) 0.4 (8) 0.5 (26) 0.2 (7) Total lignin 17.2 (100) 11.1 (100) 5.3 (100) 1.9 (100) 2.7 (100) Tricin (T) 3.1 (18) 2.7 (25) 2.0 (38) 3.0 (154) 1.4 (50) p-Coumaric acid (PCA) 1.4 (8) 0.8 (7) 0.3 (6) 0.3 (13) 0 Ferulic acid (FA) 2.6 (15) 0.3 (3) 0 0 0 Total aromatics 24.2 15.0 7.7 5.1 4.1 Sugar units 75.8 85.1 92.3 94.9 96.0 Total 100 100 100 100 100 Lignin S/G ratio 0.41 0.53 0.89 1.02 1.05 -Side chains and end groups b : β-O-4′ethers (A) 76 (59) 91 (59) 100 (16) 29 (12) 0 Phenylcoumarans (B)6(5)0 0 0 0 Resinols (C)10(8)0000 Cinnamyl end groups (I) 8 (6) 9 (6) 0 71 (30) 0 Total 100 (78) 100 (65) 100 (16) 100 (42) 0 Milled wheat straw was treated with 13 or 65 U g −1 of laccase (Lac13 and Lac65) in combination with 5, 10, or 20 % HBT (HBT5, HBT10, and HBT20, respectively) and compared with laccase alone and with a control without enzyme (followed in all cases by an extraction with alkaline peroxide) using HSQC NMR (Figs. 3and 4) a Sample composition provides the molar amount of normal (S, G, and H) and C α -oxidized (S' and G') lignin units, S/G ratio, tricin (T), p-coumaric acid (PCA), and ferulic acid (FA) from integration of aromatic signals and sugar units from integration of anomeric carbon signals, in the HSQC spectra (parentheses, values referred to total lignin); total lignin (italics)= S+S'+G+G'+H, total aromatics (italics)= total lignin+T+PCA+FA b The percentages of lignin side chains forming different substructure (A-C) and cinnamyl alcohol end groups are provided from integration of aliphatic signals in the HSQC spectra (parentheses, values referred to total lignin) 928 Bioenerg. Res. (2016) 9:917–930 ϵϬ
attaining a lignin removal up to 37 %. The delignification can be improved up to 48 % if a subsequent alkaline peroxide extraction step is combined with the enzymatic treatment. Although these lignin removal values were attained with high mediator doses, noteworthy was the lignin reduction reached (up to 38 %) in lower-dose treatments, which would be more industrially attractive. In all cases assayed, the pretreated wheat straw was hydrolyzed with higher efficiency than the untreated material releasing higher yields of glucose and xylose by using relatively low doses of cellulases. The 2D NMR spectra of whole straw samples (at the gel stage) showed the selective action of laccase-mediator on the lignin moiety, while the polysaccharide signals remained unchanged with respect to the controls. This included the breakdown of inter-unit linkages and removal of lignin S and G units, although tricin remained in the residual lignin. The presence of oxidized S and G lignin units in the pretreated wheat straw provides evidence for a C α -oxidation mechanism (and β-O-4′ cleavage) of lignin degradation even in the treatments with laccase alone. It should be pointed out that although promising results have been obtained in the present work with the laccase-mediator pretreatment of wheat straw, further optimization of the treatment including the increase in solid loading and the decrease of the laccase-mediator doses should be necessary to approach industrial conditions. Acknowledgments This study was funded by the INDOX EU-project (KBBE-2013-7-613549); the LIGNOCELL, LIGNIN, NOESIS, and BIORENZYMERY Spanish MICINN (co-financed by FEDER funds) projects (AGL2011-25379, CTQ2014-60764-JIN, BIO2014-56388 R and AGL2014-53730-R); and the CSIC (201440E097) Project. A.P. thanks the Spanish MINECO for a FPI fellowship. A. Lomascolo and E. 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Kirk TK, Farrell RL (1987) Enzymatic “combustion”: the microbial degradation of lignin. Annu Rev Microbiol 41:465–505 44. Ruiz-Dueñas FJ, Martínez AT (2009) Microbial degradation of lignin: how a bulky recalcitrant polymer is efficiently recycled in nature and how we can take advantage of this. Microb Biotechnol 2:164–177 45. Kawai S, Nakagawa M, Ohashi H (2002) Degradation mechanisms ofanonphenol ic β-O-4 lignin model dimer by Trametes versicolor laccase in the presence of 1-hydroxybenzotriazole. Enzym Microb Technol 30:482–489 46. Cantarella G, Galli C, Gentili P (2003) Free radical versus electrontransfer routes of oxidation of hydrocarbons by laccase-mediator systems. Catalytic and stoichiometric procedures. J Mol Catal B- Enzym 22:135–144 47. Areskogh D, Li J, Nousiainen P, Gellerstedt G, Sipila J, Henriksson G (2010) Oxidative polymerisation of models for phenolic lignin end-groups by laccase. Holzforschung 64:21–34 48. Eriksson K-EL, Blanchette RA, Ander P (1990) Microbial and enzymatic degradation of wood components. Springer-Verlag, Berlin 49. Gierer J, Ljunggren S (1979) Reactions of lignins during sulfate pulping.16. Kinetics of the cleavage of β-aryl ether linkages in structures containing carbonyl groups. Svensk Papperstidning- Nordisk Cellulosa 82:71–81 930 Bioenerg. Res. (2016) 9:917–930 ϵϮ
Publicaciones V PUBLICACIÓN Ϯ: Rencoret J., Pereira A., del Río J.C., Martínez A.T., Gutiérrez A. (2017) Delignification and saccharification enhancement of sugarcane byproducts by a laccase-based pretreatment. ACS Sustainable Chemistry & Engineering 5: 7145-7154. ϵϯ
Delignification and Saccharification Enhancement of Sugarcane Byproducts by a Laccase-Based Pretreatment Jorge Rencoret,* ,† Antonio Pereira, † JoseC. del Río, † Angel T. Martínez, ‡ and Ana Gutierrez † † Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, Reina Mercedes 10, E-41012 Seville, Spain ‡ Centro de Investigaciones Biologicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain ABSTRACT: Sugarcane bagasse and straw, two major agroindustrial byproducts generated by the sugarcane industry, contain significant amounts of carbohydrates that can be hydrolyzed and then converted into ethanol or other valuable compounds. However, access to them is limited by the presence of lignin, a recalcitrant polymer that protects cell-wall polysaccharides from enzymatic hydrolysis. This work demonstrates the ability of an enzymatic pretreatment, based on the laccase from Pycnoporus cinnabarinus, and 1-hydroxybenzotriazole as mediator, to remove and/or modify lignin in sugarcane bagasse and straw residues, improving their subsequent saccharification. Up to 27% and 31% decreases of relative lignin content in ground sugarcane bagasse and straw, respectively, were achieved by the laccase-mediator pretreatment followed by alkaline peroxide extraction. Moreover, the lignin removal directly correlated with improvements in enzymatic saccharification, increasing glucose releases by around 39% and 46% for bagasse and straw, respectively, compared with those of the corresponding controls. Lignin depolymerization and degradation were made evident in the 2D-NMR spectra by a significant reduction in the number of aliphatic side chains involved in the main β-O-4′and β-5′interunit linkages, together with a remarkable removal of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) lignin units as well as the associated pcoumarates and ferulates, with respect to polysaccharides. KEYWORDS: Sugarcane residues, Bioethanol, Lignocellulose, Lignin removal, Oxidoreductases, Hydrolysis, 2D-NMR ■INTRODUCTION In recent years, there has been an emerging effort to increase the production of biofuels from renewable feedstocks as a consequence of growing concerns about the depletion of fossil fuel reserves and global warming from the use of nonrenewable resources. In this context, lignocellulosic biomass represents a sustainable alternative platform to fossil resources since it is the most abundant and renewable biomass on Earth. Secondgeneration bioethanol, obtained after hydrolysis and fermentation of carbohydrates present in lignocellulosic materials, represents a real alternative to reduce the fossil fuel demand. Among the different lignocellulosic biomasses, agro-industrial lignocellulosic residues, such as sugarcane bagasse and straw, are attractive feedstocks for bioethanol production because of their high carbohydrate contents. 1 Sugarcane bagasse and straw are generated in significant amounts by the sugarcane industry, 2 around 125−140 kg 3 and 85−115 kg (dry weight), 4 respectively, per metric ton of sugarcane, and are readily available at low cost. Carbohydrates are found in lignocellulosic materials as cellulose and hemicelluloses, two major structural polymers of the plant cell-wall together with lignin. The carbohydrate fraction, especially cellulose, can be converted into fermentable reducing sugars by enzymatic hydrolysis or chemical methods. 5 The hydrolysis is usually carried out by cellulolytic enzymes, 6 and the fermentation is accomplished by using yeasts 7 or bacteria. 8 However, the accessibility of the hydrolytic enzymes to the carbohydrates in the lignocellulosic materials is hampered, to a certain extent, by the presence of lignin. Lignin is an amorphous, cross-linked, and complex aromatic polymer consisting of mainly three different phenyl-propane units linked together by different ether and carbon−carbon bonds. 9 Lignin acts as a biological cement contributing to the formation of a highly recalcitrant lignocellulosic matrix. Lignin has been shown to have a negative effect on the enzymatic hydrolysis of cell-wall polysaccharides because it strongly reduces the access of enzymes 10 and also binds to them thus reducing their activity. 11 Hence, a biomass pretreatment step is needed to partially remove lignin and break down the lignocellulose structure, making cellulose more accessible during subsequent saccharification. Given the potential of sugarcane residues for the production of second-generation bioethanol, many pretreatment processes have been developed to reduce their recalcitrance to enzymatic hydrolysis. 12 These include, among others, steam explosion, 13 organosolv, 14 liquid hot water, 15 Received: April 28, 2017 Revised: June 8, 2017 Published: July 4, 2017 Research Article pubs.acs.org/journal/ascecg © 2017 American Chemical Society 7145 DOI: 10.1021/acssuschemeng.7b01332 ACS Sustainable Chem. Eng. 2017, 5, 7145−7154 ϵϱ
ammonia fiber explosion, 16 wet oxidation, 17 alkali delignification, and dilute acid hydrolysis. 18 Biotechnology based on lignin-degrading microbes and their enzymes can contribute to the efficient and ecofriendly use of lignocellulosic feedstocks for the sustainable production of bioethanol. 19 In nature, efficient and selective lignin biodegradation is mediated mainly by white-rot fungi and certain bacteria. 20,21 The ligninolytic enzymes involved in this process are classified as peroxidases (lignin, manganese, and versatile peroxidases) and laccases. 20 Among these, laccases (phenoloxidases, EC 1.10.3.2) seem to be the most suitable enzymes for industrial applications as they can be produced on a large scale, 22 have broad substrate specificity, and utilize atmospheric oxygen as an electron acceptor to produce water. Several laccases have been shown to be capable of degrading different types of lignin, including natural 23−26 and synthetic (dehydrogenation polymer, DHP) lignins. 27 They oxidize either the minor phenolic components of lignin (less than 20%) directly (by themselves) or the phenolic and nonphenolic components, in the presence of a proper redox mediator, indirectly. As a result, radicals are generated in lignin, which can lead to bond cleavage and, consequently, to lignin depolymerization. However, little is known about the application of biological pretreatments on sugarcane residues, and almost all of them are limited to the use of lignin-degrading fungi. 28−30 These fungal pretreatments present certain advantages over the thermochemical ones, such as mild reaction conditions, higher product yields, and less energy demand; nevertheless, they require a long incubation period. This drawback can be overcome by directly using the oxidoreductase enzymes (laccases and/or peroxidases) secreted by these microorganisms for lignin degradation. The enzymatic treatments, in contrast to those that are fungal-based, require the use of previously milled lignocellulosic materials, to increase the contact surface between enzyme and substrate, although substrate sterilization is not necessary. Several studies have demonstrated the potential of the laccase-mediator system as an efficient pretreatment to remove lignin from both woody and nonwoody plant feedstocks to enhance their subsequent saccharification. 23−26 In the present study, a laccase-mediator system composed of the high-redox- potential laccase from the basidiomycete Pycnoporus cinnabarinus and 1-hydroxybenzotriazole (HBT) as mediator was investigated as a pretreatment to remove and/or modify the lignins in the sugarcane bagasse and straw residues for improved saccharification. P. cinnabarinus is one of the fungi selected for the production of high-redox-potential laccases for applications in lignocellulose biorefineries and other industrial processes. 31,32 The secretion rate, over 1.5 g L−1enzyme (in cultures containing 5 g L−1fungal biomass), is among the highest reported for a natural laccase producing fungus, and similar levels have been attained during laccase heterologous expression. Interestingly, under optimized production conditions, laccase is by far the most abundant extracellular oxidoreductase secreted by P. cinnabarinus, and therefore, crude culture filtrates could be used for lignocellulose pretreatment, without enzyme purification, as suggested for other applications. 33 ■EXPERIMENTAL SECTION Lignocellulosic Feedstocks, Enzyme, and Mediator. Sugarcane bagasse and straw were supplied by a midsized ethanol mill located in Minas Gerais state, Brazil. Sugarcane plants were harvested at the age of 6−8 months from high-performance sugarcane (Saccharum sp. hybrids) plantations. The sugarcane plants were manually collected and cleaned in the field where about one third of their weight was removed in the form of tops and leaves; this material is known as sugarcane straw or trash by the sugarcane industry. Bagasse is the solid byproduct that remains after sugarcane stalks are crushed to extract the juice. Sugarcane bagasse and straw samples were air-dried and ground using an IKA knife mill and then finely milled using a Retsch PM100 mill at 400 rpm for 6 h (with 10 min breaks after every 10 min of milling). A fungal laccase from the basidiomycete P. cinnabarinus, provided by INRA (Marseille, France), was used in this study. Its activity, measured as the initial rate during oxidation of 5 mM 2,2′-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid), ABTS (Roche Diagnostics, Mannheim, Germany), to its cation radical (ε436 29 300 M−1cm−1) in 0.1 M sodium acetate (pH 5) at 24 °C, was 525 U mL−1; the activity unit (U) was defined as the amount of enzyme oxidizing 1 μmol of ABTS per min. HBT from Sigma-Aldrich (Steinheim, Germany) was used as the redox mediator. Laccase-Mediator Treatments. The enzymatic pretreatment used to delignify the sugarcane bagasse and straw residues consisted of a sequence of four laccase-mediator treatments, using the P. cinnabarinus laccase and HBT as redox mediator, each one followed by an alkaline peroxide extraction step. These pretreatment conditions were found to efficiently remove lignin from other woody (eucalyptus) and nonwoody (elephant grass) feedstocks with important improvements in their subsequent saccharification. 23 The treatments were carried out in 200 mL pressurized bioreactors (Labomat, Mathis) placed inside a shaker bath, at 170 rpm and 50 °C, using 2 g (dry weight) of the whole biomass at 6% solid loading (w:w) in 50 mM sodium tartrate buffer (pH 4) under O2atmosphere (2 bar) for 24 h. After the enzymatic treatment, the samples were filtered and washed with 1 L of water. In a subsequent step, samples at 6% solid loading (w:w) were submitted to a peroxide alkaline extraction using 1% (w:w) NaOH and 3% (w:w) H2O2(also referred to dry weight) at 80 °C during 90 min, followed by water washing. 25 Treatments with laccase (50 U g−1) alone (without the mediator) and controls without the laccase or mediator, were also performed (followed in both cases by the corresponding alkaline peroxide extractions) for comparison. Duplicate experiments of these treatments (including control, laccase alone, and laccase-HBT) were performed to estimate the variability in biological replicates. A statistical analysis, based on ANOVA (one-way analysis of variance) and the Tukey HSD test, was carried out to compare the effects of the different enzymatic treatments on the lignin removal and on the release of glucose. Lignin content was determined as Klason lignin, in triplicate assays, according to Tappi Method T222 om-88. 34 The data from both biological and technical replicates were averaged. Saccharification of Treated Sugarcane Bagasse and Straw. The enzymatically treated sugarcane samples were hydrolyzed with a cocktail containing commercial enzymes (from Novozymes, Bagsvaerd) with cellulase [Celluclast 1.5 L; 2 FPU g−1(FPU, filter-paper unit)] and β-glucosidase (Novozym 188; 6 U g−1) activities, at 1% solid loading in 3 mL of 100 mM sodium citrate (pH 5) for 72 h at 45 °C, in a shaker bath at 140 rpm. The released glucose was determined as alditol acetate by gas chromatography (GC). 35 An HP 5890 gas chromatograph (Hewlett-Packard, Hoofddorp, The Netherlands) equipped with a split−splitless injector and a flame ionization detector was used. The injector and detector temperatures were set at 225 and 250 °C, respectively. Samples were injected in the split mode (split ratio 10:1), using a capillary column Agilent J&W DB-225 (30 m × 0.25 mm i.d. and 0.15 μmfilm thickness) and helium as the carrier gas. The oven was temperature-programmed from 220 (held for 5 min) to 230 (held for 5 min) °Cat2°C min−1. Peaks were quantified by area, and glucose was used as a standard to elaborate calibration curves. The data from three replicates were averaged. 2D-NMR Spectroscopy. For gel-state NMR sample preparation, ∼70 mg of ball-milled treated sugarcane bagasse and straw samples were transferred into 5 mm NMR tubes, and swelled in 0.75 mL of deuterated dimethyl sulfoxide (DMSO-d6), forming a gel inside the ACS Sustainable Chemistry & Engineering Research Article DOI: 10.1021/acssuschemeng.7b01332 ACS Sustainable Chem. Eng. 2017, 5, 7145−7154 7146 ϵϲ
NMR tube. 36,37 Heteronuclear single quantum coherence (HSQC) 2D-NMR experiments were run at 298 K on a Bruker AVANCE III 500 MHz spectrometer fitted with a 5 mm TCI (triple cryoprobe inverse) probe. The 2D-HSQC spectra were acquired using an adiabatic pulse sequence (Bruker standard pulse program “hsqcetgpsisp.2”), which enabled a semiquantitative analysis of the different 13C−1H correlation signals. 38 Spectra were acquired from 10 to 0 ppm in F2 (1H) using 1000 data points for an acquisition time (AQ) of 100 ms, an interscan delay (D1) of 1 s, and from 200 to 0 ppm in F1 (13C) using 256 increments of 32 scans, for a total acquisition time of 2 h 34 min. The 1JCH used was 145 Hz. Processing used typically matched Gaussian apodization in 1H (parameters LB = −0.1 and GB = 0.001) and a squared cosine bell in 13C (LB = 0.3 and GB = 0.1). The central residual DMSO peak (δC/δH, 39.5/2.49) was used as an internal reference. The 13C−1H correlation signals from the aromatic/ unsaturated region of the spectrum were used to estimate the content of lignin, p-coumarate, and ferulate, and the lignin composition in terms of H, G, S, and oxidized S (S′) and G (G′) units. The amorphous polysaccharide content was estimated by the integration of xylose and glucose anomeric signals, whereas the correlation signals in the aliphatic oxygenated region were used to determine the interunit linkage and end-unit abundances in lignin. 26 ■RESULTS AND DISCUSSION In this work, we evaluate for the first time the ability of a laccase-based enzymatic pretreatment to successfully delignify and improve the saccharification of nonchemically pretreated sugarcane residues (bagasse and straw), which present rather different lignin compositions. Bagasse is enriched in S-lignin units and uncondensed β-ether linkages, whereas straw is enriched in G-lignin units and presents more condensed linkages. 1 Furthermore, the structural modifications produced in the lignin polymer of these sugarcane byproducts during the enzymatic treatment were analyzed in situ by two-dimensional nuclear magnetic resonance (2D-NMR) at the gel state, 36,37 which provided useful information regarding the lignin degradation mechanism. Delignification of Sugarcane Bagasse and Straw with Laccase-HBT. The lignin contents (as Klason lignin) of the sugarcane bagasse and straw samples after the laccase-mediator pretreatment were determined and compared with their corresponding controls (Table 1). The lignin contents in the control samples that were processed as the full enzymatic treatment but without the presence of laccases and mediators were not modified with respect to the initial sugarcane residues. The treatments with laccase alone only decreased the lignin content of bagasse by about 4% (relative to the control), whereas a more pronounced decrease of up to 9% of the lignin content was attained in the case of straw. This low extent of degradation with laccase alone is in agreement with previous works using other lignocellulosic materials. 23−26 It is known that laccases alone are not very efficient for degrading the lignin in lignocellulosic materials because of the steric hindrance caused by their bulky molecular size and also because of their relatively low oxidation potentials, which only allow them to oxidize the minor phenolic hydroxyl groups present in lignin. However, it is important to highlight the higher extent of lignin degradation in the straw (nearly 2-fold higher), although straw only has a slightly lower lignin content than bagasse. This fact seems to suggest that the lignin composition of these materials, which is different in bagasse (S-rich lignin) and straw (G-rich lignin), 1 plays a major role in the lignin degradation with laccase alone. Previous works have shown that G-lignin units are present as phenolic units in a higher extent than the S-lignin units, which are mostly etherified, 39,40 and this would explain their higher degradation extent with laccases alone. However, the lignin content in both lignocellulosic residues significantly decreased after the enzymatic treatment using the laccase-HBT system. For sugarcane bagasse, the lignin reduction was about 27% of the initial lignin content whereas in the case of sugarcane straw up to 31% of lignin removal was achieved. Similar delignification degrees (ca. 32%) were obtained with elephant grass using the same enzymatic conditions, although much higher delignification degrees (nearly 50% lignin removal) could be attained with eucalyptus wood. 23 The differences in the delignification extents of elephant grass and those of eucalyptus wood were attributed to the predominance of syringyl lignin units in the latter. 23 Nevertheless, this seems not to be the case for the sugarcane residues selected for this study since sugarcane bagasse is enriched in S-lignin units 1 but presented a slightly lower delignification extent than the straw, which is enriched in G-lignin units 1 and presented a slightly higher delignification extent. These data indicate that, in the pretreatment of sugarcane residues, other structural features of the lignin polymer beyond its composition (H:G:S ratio) have to be considered to explain the obtained delignification results. In this sense, a possible reason could be related to the presence of p-coumarates acylating the γ-OH of the lignin side chains, which could hamper in some way the action of laccases. This fact would explain why sugarcane straw, with a percentage of pcoumarates of 47% (referred to lignin), is delignified to a greater extent than sugarcane bagasse, which has a higher content of p-coumarates (77%). This hypothesis would also Table 1. Percentage of the Initial Material Recovered, Lignin Content, Glucose Released by Enzymatic Hydrolysis, and in Vitro Digestibility of Sugarcane Residue Samples a sample recovery (%) lignin (%) b glucose (%) b digestibility (%) c Sugarcane Bagasse initial sugarcane bagasse 17.8 ±0.6 35.9 ±0.7 71.7 ±1.1 control d 88.5 17.5 ±0.4a 40.1 ±0.2a 70.9 ±0.3a laccase (50 U g−1) d 80.5 16.8 ±0.3a 44.2 ±0.4b 71.3 ±0.3a laccase (50 U g−1), HBT (3%) d 74.5 12.8 ±0.3b 55.8 ±0.4c 82.9 ±0.6b Sugarcane Straw initial sugarcane straw 17.0 ±0.2 34.9 ±0.2 75.4 ±0.4 control d 90.5 16.6 ±0.2a 39.2 ±0.2a 76.5 ±0.4a laccase (50 U g−1) d 82.0 15.1 ±0.1b 42.3 ±0.1b 75.0 ±0.2a laccase (50 U g−1), HBT (3%) d 75.5 11.5 ±0.3c 57.1 ±0.3c 93.0 ±0.5b a Means ±SD (standard deviation) shown were obtained from technical triplicates. Letters next to the SD, from the Tukey test, show results not significantly different from the control (a), significantly different from the control (b) and significantly different from both the control and the laccase-alone results, at the 0.05 level. b Values for the lignin content (expressed as Klason lignin) and glucose released after cellulase hydrolysis of samples treated with P. cinnabarinus laccase (50 Ug −1) and HBT (3%), followed by an alkaline peroxide extraction (Ep), are compared with values for a control without enzyme, a treatment only with laccase, and the initial sugarcane bagasse and straw samples. c Digestibility values were calculated on the basis of the total glucan content in the initial sugarcane residues and the percentages of released glucose, with consideration in each case for the percentage of recovery. d Enzymatic/Ep pretreated (4 cycles). ACS Sustainable Chemistry & Engineering Research Article DOI: 10.1021/acssuschemeng.7b01332 ACS Sustainable Chem. Eng. 2017, 5, 7145−7154 7147 ϵϳ
explain that the lignin removal attained after pretreatment of elephant grass (32%) 23 was close to those achieved in the case of sugarcane bagasse and straw pretreatment since elephant grass lignin is also γ-p-coumaroylated (∼40%). 41 Finally, in the case of eucalypt wood, whose lignin is not acylated at the γ-OH of the side chains, 42 the pretreatment should be more effective, and this would explain why the attained lignin removal was nearly 50%. Enzymatic Hydrolysis of Sugarcane Bagasse and Straw Pretreated with Laccase-HBT. The sugarcane bagasse and straw samples treated with laccase-HBT and those treated with laccase alone (without mediator), as well as the control samples, were hydrolyzed (72 h) using low doses of cellulases (2 FPU g−1) and β-glucosidase (6 U g−1), 23 and the released glucose was quantified as alditol acetate by GC. The saccharification results (Table 1) indicate a direct correlation between the lignin removal and the increase in glucose yield obtained by enzymatic hydrolysis. After the treatment with laccase-HBT, relative glucose releases improved up to 39% and 46%, for bagasse and straw, respectively, with respect to the control samples. Interestingly, enzymatic treatments with laccase alone also showed a positive effect in the subsequent enzymatic saccharification, with improvements in glucose release around 10% for both bagasse and straw. This positive effect of the laccase alone has also been observed during the enzymatic pretreatment of wheat straw, 26 and is attributed to both the delignification of the phenolic lignin moiety and the reduction of the nonspecific bindings of cellulases to lignin, Figure 1. HSQC NMR spectra of sugarcane bagasse after laccase-mediator treatment and a subsequent alkaline peroxide extraction (4 cycles). Expanded aliphatic oxygenated (δH/δC, 2.5−6.0 and 50−110 ppm; top) and aromatic (δH/δC, 5.8−8.2 and 90−150 ppm; bottom) regions of the HSQC NMR spectra of sugarcane bagasse treated with P. cinnabarinus laccase-HBT: (a, d) control without enzyme, (b, e) 50 U g−1enzyme, and (c, f) 50 U g−1enzyme and 3% HBT. See Table 2 for lignin signal assignments, Figure 3 for the main lignin structures identified, and Table 3 for quantification of these lignin structures. Carbohydrate signals are also observed mainly corresponding to C1−C5in normal (X1−X5) and acetylated (X′1−X′5) xylan units (anomeric glucose and arabinose signals were also identified: G1and Ar1). Unassigned signals are in gray, including signals from the enzyme and the mediator. ACS Sustainable Chemistry & Engineering Research Article DOI: 10.1021/acssuschemeng.7b01332 ACS Sustainable Chem. Eng. 2017, 5, 7145−7154 7148 ϵϴ
which make them inactive. 43 Moreover, a treatment with laccase alone could also modify the lignocellulosic substrate surface by increasing the amounts of carboxylic acids, reducing in this way the nonspecific adsorption of negatively charged cellulases. 44 A possibility for the industrial implementation and environmental feasibility of the laccase pretreatments would be the use of natural phenolic mediators instead of synthetic mediators, 45 as already proposed for paper pulp delignification. 46 2D-NMR Analysis of Sugarcane Bagasse and Straw after Laccase-HBT Pretreatment. The main structural modifications produced in sugarcane bagasse and straw by the laccase-mediator treatment were studied by 2D-NMR of whole cell-walls at the gel state, according to the previously developed methodology. 36,37 This technique allows for the in situ structural characterization of lignins, without the need of prior lignin isolation that sometimes results in alterations of the lignin polymer structure, and carbohydrates in the cell-wall. Finely ball-milled samples were swelled in DMSO-d6to form a gel inside the NMR tube and then were analyzed by HSQC 2D-NMR experiments. 36,37 The spectra of enzymatically pretreated (with laccase-HBT and laccase alone) sugarcane bagasse and straw samples (including the respective control samples) are shown in Figures 1 and 2. The aliphatic oxygenated region of the spectra (δH/δC, 2.5−6.0 and 50− 110) shows correlation signals from lignin (side chains and methoxyl groups) and carbohydrates (mainly from hemicelluloses and noncrystalline cellulose), whereas the aromatic/ unsaturated region of the spectra (δH/δC, 5.8−8.2 and 90−150) includes signals from lignin units (H, G, S) as well as signals from the associated p-coumarates and ferulates. The lignin and carbohydrate cross-signals assigned in the HSQC spectra are listed in Table 2, and the main lignin units and substructures are depicted in Figure 3. The structural characteristics of the Figure 2. HSQC NMR spectra of sugarcane straw after laccase-mediator treatment and a subsequent alkaline peroxide extraction (4 cycles). Expanded aliphatic oxygenated (top) and aromatic (bottom) regions of the HSQC NMR spectra of sugarcane straw samples after treatment with laccase-HBT followed by an alkaline peroxide extraction: (a, d) control without enzyme, (b, e) 50 U g−1enzyme, and (c, f) 50 U g−1enzyme and 3% HBT. See the Figure 1 legend for additional information. ACS Sustainable Chemistry & Engineering Research Article DOI: 10.1021/acssuschemeng.7b01332 ACS Sustainable Chem. Eng. 2017, 5, 7145−7154 7149 ϵϵ
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Short title: Enzymatic pretreatment of P. fortunei A commercial laccase-mediator system to delignify and improve saccharification of the fast-growing Paulownia fortunei Jorge Rencoret1,*, Antonio Pereira1, Gisela Marques1, José Carlos del Río1, Ángel T. Martínez2 and Ana Gutiérrez1 1Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, E-41012-Seville, Spain 2Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040-Madrid, Spain * Corresponding author: (Tel: +34 954624711; E-mail: [email protected]c.es) Abstract: It was demonstrated for the first time that a laccase-based enzymatic pretreatment is able to delignify fast-growing paulownia species. The treatment was performed with a commercial low-redox potential laccase isolated from Myceliophthora thermophila and methyl syringate (MeS) as a natural phenolic mediator. Up to 24% lignin removal was attained by the laccase-mediator treatment (L/MeS), followed by alkaline peroxide extraction in a multistage sequence. The reduction in lignin content was accompanied by a significant improvement in the subsequent enzymatic saccharification ending up in 38% glucose and 34% xylose yields. The structural modifications of the lignin were analyzed in situ by 2D- NMR spectroscopy. A considerable removal of guaiacyl and syringyl lignin units with respect to the carbohydrate signals was visible as well as the cleavage of ȕ-O-4މ, ȕ-5މ and ȕ-ȕމ linkages leading to elevated amounts of CĮ-oxidized guaiacyl and syringyl units. The presence of oxidized lignin compounds in the filtrates of the enzymatic treatments ņ such as vanillin, vanillic acid, syringaldehyde and syringic acid ņ conclusively demonstrates the ability of L/MeS treatment to depolymerize the lignin in paulownia wood. Keywords: pretreatment; paulownia; lignin; 2D-NMR; laccase; saccharification ϭϬϵ
Introduction Paulownia genus comprises of nine species and a few hybrids native to China and East Asian (Zhu et al. 1986), which are grown commercially for the production of hardwood timber (Bergmann 1998), while Paulownia is a fast-growing tree with a high biomass production (Jiménez et al. 2005). Its properties such a lightweight, strength, insulation, fast drying, high ignition point and rot resistance makes it popular for house construction and furniture making. It was also investigated as a raw material for the production of chemical pulp (Jiménez et al. 2005; Caparrós et al. 2007, 2008). Paulownia fortunei is the most suitable species to this purpose (Rai et al. 2000). P. fortunei shows extraordinary high growth rates under suitable conditions (Ede et al. 1997), reaching up to 15-20 m high in only 5-7 years, and annual productions as high as 150 t ha-1 y-1 (Jiménez et al. 2005). Paulownia also has a potential use as energy crop for the production of bioethanol (Ye et al. 2016; Domínguez et al. 2017) by enzymatic hydrolysis (Chandra et al. 2007) and subsequent fermentation by yeasts (Park et al. 2013) or bacteria (Ng et al. 1981). To this purpose, the cross-linked macro-molecular assembly of the cell wall must be submitted to a pretreatment (Chen 2014). Especially lignins limit the enzymatic hydrolysis by steric hindrance of the enzymes access to the polysaccharides and their inactivation (Kumar and Wyman 2009; Rahikainen et al. 2013). Paulownia wood contains ~24% lignin, which is composed of guaiacyl (G) and syringyl (S) units with an S/G ratio of 0.66 (Rencoret et al. 2009a). Different physical and chemical pretreatments have been proposed for a better saccharification of paulownia wood, such as dilute acid, alkali, ultrasonic-assisted alkali treatments (Ye et al. 2015, 2016), autohydrolysis (Domínguez et al. 2017), and steam explosion (Radeva et al. 2012). Biological pretreatments are also possible via ligninolytic fungi or their enzymes, but these methods have not yet been investigated on paulownia. ϭϭϬ
The aim of the present study is to evaluate the pretreatment of paulownia wood by commercially available laccase-mediator (L/M) system to improve the subsequent saccharification. The thermostable laccase from the fungus M. thermophila will be applied from Novozymes (Bagsvaerd, Denmark) that has been cloned and expressed in Aspergillus oryzae (Xu et al. 1996; Berka et al. 1997). Methyl syringate (MeS) will serve as mediator, which is obtained from syringic acid present in pulp and paper side-streams (Rosado et al. 2012). MeS is cheaper and less toxic than synthetic mediators such as HBT, violuric acid or ABTS. The L/MeS treatment was proved to be highly effective on eucalypt wood (Rico et al. 2014), which contains a syringyl-rich lignin (Rencoret et al. 2008) that is easier to degrade under alkaline conditions (González-Vila et al. 1999; Shimizu et al. 2017). The modification of the structure of the lignin polymer in the pretreated paulownia will be monitored by 2D- NMR spectroscopy of the whole sample at the gel state (Kim et al. 2008; Rencoret et al. 2009b), and the effect of the L/MeS treatment on the saccharification yield will be reported. Material and methods Paulownia wood, enzymes and mediator: 3-year-old P. fortunei trees were provided by the University of Huelva (Spain). The wood was manually debarked, chipped, air-dried and ground in a knife mill IKA MF1O to pass 1 mm screen, and then finely milled in a planetary mill Retsch PM100 at 400 rpm for 2 h (including pause times to prevent sample heating), using a 500 mL agate jar and agate balls (20 × 20 mm). The commercial (recombinant) fungal laccase from M. thermophila (Novozym 51003) was supplied by Novozymes. The enzymatic activity was measured as initial velocity during oxidation of 5 mM ABTS from Roche to its cation radical (İ436 29300 M-1 cm-1) in 0.1 M sodium acetate (pH 5) at 24°C. The laccase activity was 1000 U mL-1. One activity unit (U) is defined as the amount of enzyme transforming 1 ȝmol of ABTS per min. MeS (methyl 4-hydroxy-3,5-dimethoxybenzoate from Alfa Aesar (Karlsruhe, Germany) served as the redox mediator. ϭϭϭ
L/MeS treatments: A sequence of four L/MeS treatments was applied, each one followed by an alkaline peroxide extraction step. Laccase doses of 50 U g-1 were assayed, together with 3% MeS (% is b.o. dry wood). Paulownia samples (4 g) at 6% consistency (w:w) in 50 mM NaH2PO4 (pH 6.5) were placed into 200 mL pressurized bioreactors (Labomat, Mathis, Switzerland) and treated under O2 atmosphere (2 bar), in a thermostatic shaker (adjusted at 50°C and 190 rpm), for 24 h. Then, samples were filtered through a Büchner funnel and washed with 1 L of water. Subsequently, samples at 6% consistency (w:w) were submitted to an alkaline peroxide extraction wit 1% (w:w) NaOH and 3% (w:w) H2O2 at 80°C for 90 min in a thermostatic shaker at 140 rpm, followed by water washing. Treatments with laccase alone (50 U g-1), without mediator, and controls without laccase and mediator were also performed. A control treatment with mediator alone was not included in view of the negative results of previous studies. The Klason lignin contents (TAPPI Method T222 om-88, 2004) were corrected for ash. Saccharification of pretreated samples: After L/MeS treatment, the samples were hydrolyzed in an enzyme cocktail of commercial enzymes (Novozymes) with cellulase (Celluclast 1.5 L; 2 FPU g-1) and ȕ-glucosidase (Novozym 188; 6 U g-1) at 1% solid loading in 3 mL of 100 mM sodium citrate (pH 5) for 72 h at 45°C, in a shaker bath at 140 rpm. The released monosaccharides were determined as alditol acetate derivatives by GC (Selvendran et al. 1979) on an HP 5890 instrument (Hewlett-Packard, Hoofddorp, The Netherlands), as previously described (Rencoret et al. 2017). Chromatographic peaks were quantified by area, and different standards (including glucose and xylose, among others) were used to elaborate calibration curves. Duplicate experiments were performed in terms of of L/MeS treatment (including control, laccase and laccase-MeS) and glucose and xylose release, and Klason lignin contents were analysed in triplicate measurements. At the 95% confidence level, the enzymatic experiments are smaller than the differences found between the control, laccase alone, and L/MeS treatments. Moreover, ANOVA experiments between subjects were ϭϭϮ
performed. Post hoc pairwise comparisons, using the Tukey HSD test, were also performed. The data from both enzymatic and technical replicates were averaged. 2D-NMR analyses: The 2D HSQC NMR experiments were performed at the gel state, which is an in situ analysis of the whole cell-wall (Kim et al. 2008, Rencoret et al. 2009b). This approach does not require a previous lignin isolation and avoids possible alterations and material losses during the isolation process. These experiments provided also structural information on the hemicelluloses in the cell wall. 70 mg of ball-milled samples (and filtrate samples) were transferred into 5-mm NMR tubes, and swelled in 1 mL of DMSO-d6, forming a gel inside the tube (Kim et al. 2008; Rencoret et al. 2009b). Heteronuclear single quantum coherence (HSQC) 2D-NMR spectra were recorded at 300 K on a Bruker AVANCE III 500 MHz spectrometer equipped with a 5 mm TCI gradient cryoprobe with inverse geometry. An adiabatic HSQC pulse sequence (Bruker standard ‘hsqcetgpsisp.2’), which enabled a semiquantitative analysis of the different 1H-13C correlation signals (Kupþe and Freeman, 2007), was utilized. HSQC spectra were acquired from 10 to 0 ppm (5000 Hz) in F2 (1H) using 1000 data points for an acquisition time (AQ) of 100 ms, an interscan delay (D1) of 1 s, and from 200 to 0 ppm (25,168) in F1 (13C) using 256 increments of 32 scan, for a total acquisition time of 2 h 34 min. The 1JCH used was 145 Hz. Processing used typical matched Gaussian apodization in 1H and a squared cosine bell in 13C. The central solvent peak was used as an internal reference (įC/įH 39.5/2.49). Lignin and carbohydrate correlation signals in the HSQC spectra were assigned by comparison with the literature (Rencoret et al. 2009a; 2009b; Kim et al. 2014). The 1H-13C correlation signals from the aromatic region of the spectrum were used to estimate the content of lignin (relative to the content of amorphous carbohydrates, estimated from the anomeric xylose and glucose signals), and the lignin composition in terms of G, S and oxidized S (Sƍ) and G (Gƍ) units. The CD-HD correlation signals in the aliphatic-oxygenated region were used to estimate the abundance of the various lignin inter-unit linkages whereas the CȖ-HȖ correlation signals were used to estimate the ϭϭϯ
abundance of the cinnamyl alcohol end-units. Likewise, S2,6 (and Sƍ2,6) and G2 (and Gƍ2) signals were used to estimate the relative abundances of the aromatic units – as signals S2,6 and Sƍ2,6 involve two proton-carbon pairs, their volume integrals were halved. The content of CĮ-oxidized S-lignin units in the HSQC spectrum of paulownia treated with laccase and mediator was corrected for the contribution of MeS to the signal at įC/įH 106.2/7.20, which was estimated from the integral of its characteristic signal at įC/įH 52/3.8 ppm. Analyses of enzymatic filtrates: The filtrates obtained after each enzymatic treatment were combined (from the four cycles), lyophilized and subsequently extracted with chloroform by sonication (20 min). The solution was centrifuged (10000 rpm for 25 min) and the supernatant, which contains low molecular weight lignin-derived compounds, was then collected by decantation. The extraction process was repeated three times, using fresh chloroform each time and the supernatants were combined. The chloroform was removed by rotary evaporation at 40°C and the compounds were then silylated with bis(trimethylsilyl)trifluoroacetamide (BSTFA)-pyridine (2:1 v/v) and analyzed by GC-MS on a Varian Saturn 4000 equipment. The GC column: 15 m × 0.25 mm i.d., 0.1 ȝm film thickness, DB5-HT (J&W Scientific). The temperature program: 50°C ĺ 90°C (30°C min-1), holding time 2 min, ĺ 250°C (8°C min-1), holding time 2 min. The injector and transfer line temperatures were 250°C and 300°C, respectively. He was the carrier gas (2 mL min-1). The chloroform-insoluble fractions of the filtrates, which contain lignin fragment with higher molecular weights, were analyzed by 2D-NMR under the same conditions as described above. Result and discussion Delignification of paulownia wood by the enzymatic pretreatment The Klason lignin content of the paulownia control, processed as the full enzymatic treatment but without laccase and mediator, was only slightly reduced from 23.8 to 22.0% with respect to the initial paulownia lignin content (Table 1). This is the effect of alkaline peroxide ϭϭϰ
extractions. The treatment with laccase alone only resulted in a 5% lignin reduction compared to the control sample. This low efficiency of laccase alone is well known (Lai, 1992). The pretreatment with L/MeS, however, resulted in a lignin decrement up to 24%, with respect to the control, but this delignification degree is lower than reported about eucalypt wood treatment under the same experimental conditions (47% lignin removal). This difference may be due to the high S-units content of eucalypt wood lignin compared to the G-units-rich and condensed paulownia lignin. Saccharification of pretreated paulownia The percentages of glucose and xylose released by saccharification of treated paulownia samples are listed in Table 1. The saccharification results indicate that there is a direct correlation between the lignin removal and the higher glucose and xylose yields. The pretreatment with laccase alone enhanced the glucose and xylose yields up to 6% and 7%, respectively, but the L/MeS treatment led to glucose and xylose increase up to 38% and 34%, respectively. Rico et al. (2014) found similar glucose (41%) and xylose (37%) yield increments with eucalypt wood after L/MeS under the same experimental conditions. 2D-NMR analysis The full HSQC NMR spectra of the samples, including those resulting from the treatment with L/MeS, L alone, and the corresponding control are shown in Figure 1. A pronounced lignin removal occurred after L/MeS pretreatment, as evidenced by the lower lignin signals (colored in red) after the treatments, in comparison with the signals from carbohydrates (in cyan color) that remained mostly unchanged (Figure 1c), although some lignin degradation can also be seen in the spectrum of L treated samples (Figure 1b). More detailed information was gained from these HSQC spectra after increasing their intensities and analyzing the aliphatic oxygenated and the aromatic regions individually (Figure 2). The aliphatic ϭϭϱ
FIGURE LEGENDS Figure 1. 2D-HSQC NMR spectra of paulownia after laccase-mediator treatments followed by alkaline peroxide extractions (4 cycles): Control without enzyme (a), treated with 50 U g-1 laccase (b) and treated with 50 U g-1 laccase and 3% MeS (c). Correlation signals in reddishbrown color largely correspond to lignin, whereas signals in cyan color belong to carbohydrates. Figure 2. Expanded aliphatic oxygenated (įC/įH 50-110/2.5-6.0, top) and aromatic (įC/įH 100-125/6.0-8.0, bottom) regions of the 2D-HSQC NMR spectra of paulownia samples; Control without enzyme (a, d), treated with 50 U g-1 enzyme (b, e) and treated with 50 U g-1 enzyme and 3% MeS. The lignin and carbohydrate signal assignments are listed in Table 2 and the lignin structures identified are depicted in Figure 3. Correlation signals of MeS in the HSQC spectrum from laccase-mediator treated paulownia are observed at įC/įH 52.1/3.82 (C/H in MeS) and įC/įH 106.4/7.20 (C2/H2 and C6/H6 in the aromatic ring). Figure 3. Main lignin units and substructures identified in the 2D-HSQC spectra of treated paulownia samples. A) ȕO4ƍ alkyl-aryl ether structures; Aox) ȕO4ƍ structures with CĮ- oxidized; B) ȕ5ƍ phenylcoumaran structures; C) ȕȕƍ resinol structures; I) cinnamyl alcohol end-group; G) guaiacyl unit; S) syringyl unit; Gƍ) CĮ-oxidized G unit; Sƍ) CĮ-oxidized S unit; MeS) methyl syringate. Figure 4. Total ion chromatograms of the chloroform-soluble fraction of filtrates obtained after the enzymatic pretreatment of paulownia with laccase–mediator (C), laccase alone (B) and the corresponding control (A). The lignin-derived simple compounds identified are depicted (a-e). MeS refers to methyl syringate whereas the peak with asterisk mark (*) refers to contamination peak from methyl syringate. Figure 5. HSQC NMR spectra of lignin compounds in the chloroform-insoluble fraction of filtrates from enzymatic treatment of paulownia with laccase–mediator (c), laccase alone (b) and the corresponding control (a). See Figure 3 for the main lignin-derived structures identified. ϭϮϮ
Table 1. Laccase-mediator treatment of paulownia wood (material recovery and final lignin content) and subsequent Celluclast hydrolysis (amount of glucose and xylose per 100 g of sample)* Samples 1) Laccase-mediator treatment 2) Celluclast hydrolysis (2 FPU) Recovery (%) Lignin (%) Glucose (%) Xylose (%) Initial paulownia 100 (4 g) 23.8 ± 0.2 31.5 ± 0.3 6.7 ± 0.1 Control * 83 (3.32 g) 22.0 ± 0.1a 37.3 ± 0.2a 7.3 ± 0.1a Laccase (50 U·g- 1 ) † 80 (3.21 g) 20.9 ± 0.3a 39.4 ± 0.4 b 7.8 ± 0.1a Laccase (50 U·g- 1 )-MeS (3%) † 76 (3.03 g) 16.7 ± 0.2 b 51.3 ± 0.4c 9.8 ± 0.3 b *Means ± StD presented were obtained from triplicate determination. Letters next to the StD, from the Tukey test, show: no significant differences to the control (a), significant differences (b) and significantly different from both the control and the L- alone results (c), at the 0.05 level. *followed by alkaline-peroxide extraction (4 cycles). ϭϮϯ
Table 2. Assignments of the 1H/13C correlation signals in the HSQC spectra of the whole cellwalls from treated paulownia. Label įC/įH (ppm) Assignment Lignin signals Bȕ 53.1/3.43 Cȕ/Hȕ in phenylcoumaran substructures (B) Cȕ 53.4/3.04 Cȕ/Hȕ in resinols substructures (C) MeO 55.5/3.72 C/H in methoxyls AȖ 59.6 /3.37 and 3.71 CȖ/HȖ in ȕ–O–4މ substructures (A) IȖ 61.3/4.09 CȖ/HȖ in cinnamyl alcohol end-groups (I) BȖ 62.6/3.69 CȖ/HȖ in phenylcoumaran substructures (B) AĮ(G) 70.8/4.73 CĮ/HĮ in ȕ–O–4މ substructures (A) linked to a G-unit CȖ 70.9/4.17 and 3.81 CȖ/HȖ in resinols substructures (C) AĮ(S) 71.8/4.83 CĮ/HĮ in ȕ–O–4މ substructures (A) linked to a S-unit Aoxȕ 83.0/5.20 Cȕ/Hȕ in Į-oxidized ȕ–O–4މ substructures (Aox) Aȕ(G) 83.6/4.26 Cȕ/Hȕ in ȕ–O–4މ substructures linked (A) to a G unit CĮ 84.7/4.63 CĮ/HĮ in resinols substructures (C) Aȕ(S) 85.8/4.08 Cȕ/Hȕ in ȕ–O–4މ substructures linked (A) to a S unit BĮ 86.7/5.42 CĮ/HĮ in phenylcoumaran substructures (B) S2,6 103.8/6.65 C2/H2 and C6/H6 in etherified syringyl units (S) Sމ2,6 106.2/7.20 C2/H2 and C6/H6 in Į-oxidized syringyl units (Sމ) G2 110.9/6.93 C2/H2 in guaiacyl units (G) Gމ2 111.7/7.42 C2/H2 in Į-oxidized guaiacyl units (Gމ) G5/G6 114.9/6.67 and 6.85 118.8/6.76 C5/H5 and C6/H6 in guaiacyl units (G) Gމ5 115.0/6.94 C5/H5 in Į-oxidized guaiacyl units (Gމ) Gމ6 122.7/7.54 C6/H6 in Į-oxidized guaiacyl units (Gމ) Carbohydrate signals X5 63.0/3.16 and 3.87 C5/H5 in xylopyranose units X2 72.5/3.03 C2/H2 in xylopyranose units Xމ2 73.0/4.46 C2/H2 in 2-O-acetylated xylopyranose units X3 73.9/3.24 C3/H3 in xylopyranose units Xމ3 74.7/4.79 C3/H3 in 3-O-acetylated xylopyranose units X4 75.3/3.49 C4/H4 in xylopyranose units U4 81.0/3.07 C4/H4 in 4-O-methyl-Į-D-glucuronic acid ĮX1(R) 91.9/4.88 C1/H1 in Į-D-xylopyranoside (R) [Į-D-glucopyranoside (R)] ȕX1(R) 96.5/4.31 C1/H1 in ȕ-D-xylopyranoside (R) [ȕ-D-glucopyranoside (R)] U1 97.1/5.15 C1/H1 in 4-O-methyl-Į-D-glucuronic acid Xމ1 99.4/4.48 C1/H1 in 2-O-acetylated xylopyranose units X1 101.5/4.26 C1/H1 in xylopyranose units Gl1 102.9/4.16 C1/H1 in glucopyranose units ϭϮϰ
Table 3. Semiquantitative NMR analysis of paulownia treated with M. thermophila laccase (50 U·g-1) and MeS (3%), and laccase alone, compared with control without enzyme and mediator, including sample composition and linkages/end-groups, from HSQC spectra in Figure 2. Sample composition* Control Laccase L/MeS Syringyl lignin units (S) 15.6 (37) 13.9 (42) 9.3 (36) CĮ-oxidized S units (Sƍ) 2.6 (6) 5.7 (17) 5.7 (22) § Guaiacyl lignin units (G) 23.5 (56) 11.6 (35) 6.2 (24) CĮ-oxidized G units (Gƍ) 0 (0) 2.0 (6) 4.7 (18) Total lignin 41.7 (100) 33.2 (100) 25.9 (100) Sugar units 58.3 66.8 74.1 Total 100 100 100 Lignin S/G ratio 0.8 1.4 1.4 Linkages and end groups#: ȕ-O-4ƍ ethers (A) 51 (81) 39 (95) 22 (69) ȕ-O-4ƍ ethers (A) CĮ oxidized 0 (0) 0 (0) 9 (30) Phenylcoumarans (B) 7 (11) 0 (0) 0 (0) Resinols (C) 4 (6) 2 (5) 1 (4) Cinnamyl end-groups (I) 1 (2) 0 (0) 0 (0) Total 64 (100) 41 (100) 31 (100) *Sample composition represents the molar amount of normal (H, G, and S) and CĮ-oxidized (Gƍ and Sƍ) lignin units, and sugar units (mainly xylose and glucose) from the integration of anomeric carbon signals (relative percentages of lignin units are shown in parentheses). §The contribution of MeS was subsracted. #The percentages of lignin linkages involved in substructures A, B and C, and cinnamyl alcohol end-groups (I) are referred to the total aromatic (G + Gƍ+ S + Sƍ) lignin units (% relative to 100 linkages/end-groups are provided in parentheses) obtained from integration of aliphatic oxygenated signals. ϭϮϱ
60 70 80 90 100 ppm 110 120 34567 a) 60 70 80 90 100 ppm 110 120 34567 b) 60 70 80 90 100 ppm 110 120 34567 c) Figure 1 Lignin Carbohydrates Lignin Carbohydrates Lignin Carbohydrates ϭϮϲ
CĮ Cȕ AĮ(G) MeO Aȕ(S) AȖ Bȕ X1 G1 BĮ X5X5 BȖ CȖ X2 X3 X4 AĮ(S) Aȕ(G) 5.5 5.0 4.5 4.0 3.5 3.0 60 70 80 90 100 ppm S2,6 G2 Sƍ2,6 G5/6 G6 105 110 115 120 7.5 6.0 6.5 ppm AĮ(G) AĮ(S) Aȕ(S) Aȕ(G) CĮ X1 G1 X2 X3 X4 MeO AȖ Cȕ X5X5 5.5 5.0 4.5 4.0 3.5 3.0 60 70 80 90 100 ppm X1 G1 Aȕ(S) Aoxȕ AĮ(S) X2 X3 X4 Xƍ1 Xƍ2 Xƍ3 AȖ X5X5 MeO 5.5 5.0 4.5 4.0 3.5 3.0 60 70 80 90 100 ppm S2,6 G2 Sƍ2,6 G5/6 G6 Gƍ2 Gƍ6 105 110 115 120 7.5 6.0 6.5 ppm S2,6 G2 Sƍ2,6(+MeS2,6) G5/6 G6 Gƍ2 Gƍ6 Gƍ5 105 110 115 120 7.5 6.0 6.5 ppm a) b) c) d) e) f) ĮX1(R) ȕX1(R) U1 ĮX1(R) ȕX1(R) U1 ĮX1(R) ȕX1(R) U1 U4U4 U4 Figure 2 MeS IȖ ϭϮϳ
Figure 3 A HO O HO OMe OMe O Įȕƍ Ȗ O HO OMeO OMe ƍ B Į ȕ Ȗ O O OMe O O OMe C Į ȕ Ȗ Įƍ ȕƍ Ȗƍ OMe O OH G 26 5 Į O OMe OH MeO S 26 Į O OMe OR MeO 6ƍ 26 Į Aox OO HO OMe OMe O Įȕƍ Ȗ O OMe OR *ƍ 26 Į MeS OH OMe 26 MeO O MeO I O OMe OH 26 Įȕ Ȗ (MeO) 5 ϭϮϴ
OH OMe O H a) OH OMeMeO OOH d) OH OMeMeO O H b) OH OMe OOH c) a * c MeS d b C) Laccase+MeS 109876 Retention time (min) 5 100 Relative response (%) 75 50 25 Figure 4 ac bd 109876 Retention time (min) 5 Relative response (%) 25 A) Control ~ a c b d 109876 Retention time (min) 5 Relative response (%) 25 B) Laccase alone ~ ϭϮϵ
S2,6 Sƍ2,6 G2 G5/6 G6 Gƍ2 Gƍ6 b) 105 110 115 120 125 6.66.87.07.27.47.6 S2,6 Sƍ2,6 G2 G5/6 G6 a) Aȕ(S) BĮ Aȕ(G) CĮ 82 83 84 85 86 87 5.5 5.0 4.5 4.0 105 110 115 120 125 6.66.87.07.27.47.6 Gƍ2 Gƍ6 S2,6 Sƍ2,6(+MeS2,6) G2 G6 Gƍ5 c) 105 110 115 120 125 6.66.87.07.27.47.6 G5/6 ppmppmppm Figure 5 Side-chain-linkage region Aȕ(S) Aȕ(G) CĮ 82 83 84 85 86 87 5.5 5.0 4.5 4.0 Side-chain-linkage region Aȕ(S) 82 83 84 85 86 87 5.5 5.0 4.5 4.0 Side-chain-linkage region 130 130 130 ϭϯϬ
Publicaciones V PUBLICACIÓN ϰ: Pereira A., Hoeger I.C., Ferrer A., Rencoret J., del Río J.C., Kruus K., Rahikainen J., Kellock M., Gutierrez A., Rojas O.J. (2017) Lignin films from spruce, eucalyptus, and wheat straw studied with electroacoustic and optical sensors: effect of composition and electrostatic screening on enzyme binding. Biomacromolecules 18: 1322-1332. ϭϯϭ