Função dos componentes não celulósicos na recalcitrância da parede celular

Transcrição

1 Função dos componentes não celulósicos na recalcitrância da parede celular Papel da lignina André Ferraz Departamento de Biotecnologia Escola de Engenharia de Lorena Universidade de São Paulo Lorena, SP

2 Outline - Variação natural de recalcitrância em diferentes tipos celulares - gramíneas como modelo de estudos devido a grande variabilidade de tipos celulares - Lignina como maior causador natural da recalcitrância Ref. básicas: 1. Ferraz A, Costa THF, Siqueira G, Milagres AMF 213 Mapping of cell wall components in lignified biomass as a tool to understand recalcitrance. Silva SS, Chandel AK (eds.), Biofuels in Brazil, Springer, pp Jung HG, Casler MD 26 Maize stem tissues: impact of development on cell wall degradability. Crop Sci 46: Siqueira GA, Milagres AMF, Carvalho W, Koch G, Ferraz A (211) Topochemical distribution of lignin and hydroxycinnamic acids in sugarcane cell walls and its correlation with the enzymatic hydrolysis of polysaccharides. Biotechnol Biofuels 4:1 9

3 Revendo alguns conceitos Recalcitrância: termo usualmente empregado para mencionar a dificuldade que existe para se desconstruir a parede celular vegetal Definição empregada nesta disciplina: The term refers to the restrictions imposed by the lignified cell walls to the polysaccharide hydrolysis (by enzymes or even by acids in water solution)

4 Veremos que o foco da desconstrução da parede celular é a hidrólise enzimática da celulose Pense: porque não se usa ácido para hidrolisar celulose?? Lignin and hemicelluloses are the major components limiting enzyme infiltration into the cell walls Cell wall SEM Cell wall models

5 Exemplo de solubilização de polissacarídeos em meio ácido (condições que afetam a reação) Relação sólido: líquido 1:1 Máx. de monossacarídeos esperados em solução: Xilose = 26 g/l Glicose = 42 g/l HOAc = 4,5 g/l

6 Xilose (g/l) Glicose (g/l) Glicose (g/l) Glicose (g/l) Xilose (g/l) Xilose (g/l) 2% Acid 4% Acid 6% Acid 2% Acid 4% Acid 6% Acid 2% Acid 4% Acid 6% Acid Máx. esperados: Xilose = 26 g/l Glicose = 42 g/l

7 Variação natural de recalcitrância em diferentes tipos celulares Cell wall digestibility in mature maize stalks Stem internode tissues of maize at full physiological maturity >> after in vitro degradation by rumen microbes for 24 h Jung and Casler. Crop Sci. 46: , 26.

8 Gramíneas como modelo de estudos devido a grande variabilidade de tipos celulares Cell anatomy in sugar cane vascular bundles Rind Pith

9 Cellulose conversion (%) Enzymatic hydrolysis of varied sugar cane regions from the internode Epidermis Rind ~ 3 cm Interface Pith Epidermis Hydrolysis time (h) Pith Interface Rind Epidermis Rind Interface Pith Enzymatic hydrolysis: 1 FPU/g; 2 IU 45 o C

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11 What is different in the evaluated sugar cane regions? Frequency of vascular bundles in varied fractions of a sugar cane internode Sugar cane region Number of vascular bundles/16mm 2 Pith 6 1 Interface 8 1 Rind 13 2 Epidermis Only fibers Epidermis Rind Interface Pith Volumetric and mass proportions of each sugar cane region in a sugar cane internode Ep Rind Interface Pith Sugar cane region Volumetric proportion (%) Dry mass proportion (%) Pith Interface Rind Epidermis

12 Chemical composition of varied fractions in a sugar cane internode Sugar cane region Chemical composition (%, w/w) Extractives Glucan Xylan Arabinosyl Acetyl Insoluble lignin Soluble lignin Pith Interface Rind Epidermis Hydroxycinnamic acids (%, g/1g of in natura bagasse) Released by mild-alkaline treatment Released by severe-alkaline treatment Ferulic Coumaric Sum Ferulic Coumaric Sum Pith Interface Rind Epidermis Lignina como maior causador natural da recalcitrância? Há evidências experimentais?

13 UV-microspectrophotometry was used to map lignin and hydroxycinnamic acids in the cell walls from these regions Example of a selected point (1 µm 2 ) to record the UV spectrum UV-microscope 4 x

14 Absorbance Absorbance Average spectrum from different cells in sugar cane Rind vessel fiber parenchyma Pith vessel fiber parenchyma Wavelenght (nm) Wavelenght (nm) 28 nm > aromatic ring substituted with oxygenated groups 315 nm > aromatic ring conjugated with alfa-carbonyl or alfabeta unsaturated groups >>> in grasses, correspond to ferulic and coumaric acids Siqueira et al. Biotechnol Biofuels 4:7, 211

15 Frequence of pixels Topochemical distribution of lignin.3 Frequency histograms.2 Weighed-average Absorbance.1 Absorbance values range Rind fibers Abs 28 nm =.4 Pith fibers Abs 28nm =.39 Similar lignin contents

16 Rind Parenchyma Abs 28nm =.31 Pith Parenchyma Abs 28nm =.18 Lower lignin content in pith parenchyma

17 Absrobance Absorbance Absorbance Absorbance Absorbance Absorbance Selective delignification of rind and pith regions UV-data for rind and pith regions after acetic acid/chlorite delignification vessel a RIND fiber b Wavelenght (nm) untreated 1-h treated 2-h treated cparenchyma Wavelenght (nm) untreated 1h-treated 2h-treated Wavelenght (nm) untreated 1-h treated 2-h treated PITH vessel a fiber Wavelenght (nm) untreated 1-h treated parenchyma Wavelenght (nm) untreated 1-h treated Wavelenght (nm) untreated 1-h treated

18 Lignin removal from recalcitrant rind regions UV-images from sugar cane internode cells >> after chloriteinduced delignification Cell type Untreated fibers 1-h chlorite/ acetic acid treated fibers Untreated parenchyma Rind Siqueira et al. Biotechnol Biofuels 4:7, h acetic acid/chlorite treated parenchyma

19 Cellulose conversion (%) Lignin removal (chlorite delignification) versus efficiency in the cellulose hydrolysis 7 6 Rind 7 6 Pith h-treated (7 % of lignin) 2h-treated (12 % of lignin) 1h-treated (17 % of lignin) untreated (19 % of lignin) h-treated (7 % of lignin) 1h-treated (8 % of lignin) untreated (12 % of lignin) Hydrolysis time (h) Hydrolysis time (h) Enzymatic hydrolysis: 1 FPU/g; 2 IU 45 o C Siqueira et al. Biotechnol Biofuels 4:7, 211

20 Cellulose conversion after 48 h (%) Cellulose conversion after 48h (%) Cellulose conversion after 48 h (%) Correlation of enzymatic hydrolysis efficiency with: lignin contents and UV-data in the rind R² = Total lignin content in rind (%) b vessel fiber parenchyma c vessel fiber parenchyma R² =.655 R² =.927 R² =.94 1 R² =.969 R² =.959 R² = Absorbance of rind cells at 28 nm Siqueira et al. Biotechnol Biofuels 4:7, 211 Absorbance of rind cells at 315 nm

21 Os dados sobre a recalcitrância natural de diferentes tipos celulares indicam que: Lignina deveria ser removida para facilitar a desconstrução enzimática dos polissacarídeos - Quanta lignina deveria ser removida? - Como se remove lignina?

22 Recordando What is different in the evaluated sugar cane regions? Frequency of vascular bundles in varied fractions of a sugar cane internode Sugar cane region Number of vascular bundles/16mm 2 Pith 6 1 Interface 8 1 Rind 13 2 Epidermis Only fibers Epidermis Rind Interface Pith Volumetric and mass proportions of each sugar cane region in a sugar cane internode Ep Rind Interface Pith Sugar cane region Volumetric proportion (%) Dry mass proportion (%) Pith Interface Rind Epidermis a medula representa menos do que 5% da massa total

23 Diminuindo a recalcitrância um pré-tratamento é necessário - Quanta lignina deveria ser removida? Lee et al.., Biotechnol Bioeng, 29 Selectively delignified Maple using ionic liquids Siqueira et al., Applied Energy, 213 Chlorite delignified sugarcane bagasse

24 Cellulose conversion afetr 72h of enzymatic hydrolysis (%) Cellulose conversion (%) Cellulose conversion afetr 72h of enzymatic hydrolysis (%) Direct enzymatic hydrolysis of milled sugar cane hybrids - Plantas com menos lignina são a solução?? a R² = Hydrolysis time (h) 321 mill bagasse 14 reference cultivar 1 8 Total lignin content (%) chlorite-treated reference cultivar 24.5% Intial lignin Lignin removal is necessary to reach hydrolysis levels over 7% chlorite-treated hybrid 146 (18.6% initial lignin) y = -8.11x untreated hybrids y = x Masarin et al. Biotechnol Biofuels 4:55, 211 Total lignin content (%)

25 Chen and Dixon, Nature Biotechnology, 27 Transgenic alfalfa O total de polissacarídeos hidrolisados deveria atingir cerca de 9 mg/g CWR

26 - Remoção de lignina por processos industriais de pré-tratamento são a solução?? Sulfito alcalino como agente deslignificante de bagaço de cana hemicellulose Lignin Mendes et al. Progress Biotechnol, 27:395-41, 211