Função dos componentes não celulósicos na recalcitrância da parede celular

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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

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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 2013 Mapping of cell wall components in lignified biomass as a tool to understand recalcitrance . Silva SS, Chandel AK (eds.), Biofuels in Brazil, Springer, pp. 173-202

2. Jung HG, Casler MD 2006 Maize stem tissues: impact of development on cell wall degradability. Crop Sci 46:1801–1809

3. Siqueira GA, Milagres AMF, Carvalho W, Koch G, Ferraz A (2011) Topochemical distribution of lignin and hydroxycinnamic acids in sugar-cane cell walls and its correlation with the enzymatic hydrolysis of

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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)

Recalcitrância: termo usualmente empregado para mencionar a

dificuldade que existe para se desconstruir a parede celular vegetal

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Lignin and hemicelluloses are the major components limiting

enzyme infiltration into the cell walls

Cell wall SEM

Cell wall models

Veremos que o foco da

desconstrução da parede celular é a

hidrólise enzimática da celulose

Pense: porque não se usa ácido para

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Exemplo de solubilização de polissacarídeos em meio ácido (condições que afetam a reação)

Relação sólido: líquido 1:10

Máx. de monossacarídeos esperados em solução: Xilose = 26 g/L Glicose = 42 g/L HOAc = 4,5 g/L

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Máx. esperados: Xilose = 26 g/L Glicose = 42 g/L Xi lose ( g /L ) Xil ose ( g /L ) Xil ose ( g /L ) Gli cose ( g /L ) Gli cose ( g /L ) Gli cose ( g /L ) __{2% Acid}  4% Acid ▲ 6% Acid  2% Acid  4% Acid ▲ 6% Acid  2% Acid  4% Acid ▲ 6% Acid

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Jung and Casler. Crop Sci. 46:1801-1809, 2006.

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

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Cell anatomy in sugar cane

Rind

Pith

vascular bundles

Gramíneas como modelo de estudos

devido a grande variabilidade de tipos

celulares

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Enzymatic hydrolysis of varied sugar cane regions from the internode

Rind Interface Pith Epidermis 0 20 40 60 80 100 0 20 40 60 80 C e ll u lo s e c o n v e rs io n (% ) Hydrolysis time (h) Pith Interface Rind Epidermis Epidermis Rind Interface Pith ~ 3 cm

Enzymatic hydrolysis: 10 FPU/g; 20 IU -glucosidase/g; @ 45 o_C

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What is different in the evaluated sugar cane regions?

Frequency of vascular bundles in varied fractions of a sugar cane internode

Volumetric and mass proportions of each sugar cane region in a sugar cane internode

Rind

Interface

Pith

Epidermis

Ep Rind Interface

Pith

Sugar cane region Number of vascular bundles/16mm2

Pith 6  1

Interface 8  1

Rind 13  2

Epidermis Only fibers

Sugar cane region

Volumetric proportion (%)

Dry mass proportion (%)

Pith 10 4.7  0.7

Interface 29 12  2

Rind 49 47  1

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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 _2.9 _0.2 _49.3 _{0.7 15.4} _0.2 _0.1 _0.1 _2.4_0.2 _14.8 _0.1 _2.4_0.1 Interface _3.0 _0.1 _43.8 _{0.1 18.8} _0.1 _1.4 _0.1 _3.2 _{0.1 20.4} _{0.1 2.4} _0.3 Rind _1.3 _0.1 _42.0 _{0.5 20.4} _0.2 _1.1 _0.1 _3.0 _{0.2 19.8} _{0.1 2.0} _0.3 Epidermis _3.1 _0.1 _42.4 _{0.5 19.6} _0.2 _1.2 _0.1 _2.6 _{0.2 20.4} _{0.1 1.9} _0.2

Hydroxycinnamic acids (%, g/100g of in natura bagasse)

Released by mild-alkaline treatment Released by severe-alkaline treatment Ferulic Coumaric Sum Ferulic Coumaric Sum

Pith 0.49  0.05 1.4  0.2 1.9  0.2 1.5  0.1 5.3  0.7 6.8  0.7

Interface 0.50  0.08 2.0  0.4 2.5  0.4 1.60.1 8.1  0.8 9.7  0.9

Rind 0.48  0.06 2.1  0.2 2.7  0.1 1.4  0.1 6.7  0.4 8.1  0.5

Epidermis 0.34  0.08 1.3  0.4 1.6  0.5 1.6  0.1 8.1  0.8 9.7  0.9

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UV-microspectrophotometry was used to map lignin and

hydroxycinnamic acids in the cell walls from these regions

UV-microscope

Example of a selected point (1 µm2_{) to}

record the UV spectrum

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Average spectrum from different cells in sugar cane

280 nm > aromatic ring substituted with oxygenated groups

315 nm

> aromatic ring conjugated with carbonyl or

alfa-beta unsaturated groups >>>

in grasses, correspond to ferulic

and coumaric acids

-0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 220 240 260 280 300 320 340 360 380 400 A b s o rb a n c e Wavelenght (nm) vessel fiber parenchyma -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 220 240 260 280 300 320 340 360 380 400 A b s o rb a n c e Wavelenght (nm) vessel fiber parenchyma

Rind

Pith

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Rind fibers

Abs

_{280 nm}

=0.40

Pith

fibers

Abs

_280nm

=0.39

Topochemical

distribution of lignin

Frequency histograms 0 0.1 0.2 0.3 0 0 .1 2 0 .2 0 0 .2 6 0 .3 3 0 .3 9 0 .4 6 0 .5 2 0 .5 9 0 .65 0 .72 0 .78 0 .85 0 .9 1 0 .9 7 F re q u e n ce o f p ixe ls

Absorbance values range

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Rind Parenchyma

Abs

_280nm

= 0.31

Lower lignin content in pith parenchyma

Pith

Parenchyma

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RIND -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 220 240 260 280 300 320 340 360 380 400 A b s o rb a n c e Wavelenght (nm) untreated 1-h treated 2-h treated a -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 220 240 260 280 300 320 340 360 380 400 A b s o rb a n c e Wavelenght (nm) untreated 1h-treated 2h-treated b -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 220 240 260 280 300 320 340 360 380 400 A b s ro b a n c e Wavelenght (nm) untreated 1-h treated 2-h treated c parenchyma fiber vessel PITH -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 220 240 260 280 300 320 340 360 380 400 A b s o rb a n c e Wavelenght (nm) untreated 1-h treated a -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 220 240 260 280 300 320 340 360 380 400 A b s o rb a n c e Wavelenght (nm) untreated 1-h treated -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 220 240 260 280 300 320 340 360 380 400 A b s o rb a n c e Wavelenght (nm) untreated 1-h treated vessel fiber parenchyma

UV-data for

rind and pith

regions after

acetic

acid/chlorite

delignification

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UV-images from

sugar cane

internode cells

>> after

chlorite-induced

delignification

Siqueira et al. Biotechnol Biofuels 4:7, 2011

Lignin removal

from recalcitrant

rind regions

Cell type Rind

Untreated fibers 1-h chlorite/ acetic acid treated fibers Untreated parenchyma 1-h acetic acid/chlorite treated parenchyma

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Lignin removal

(chlorite delignification)

versus

efficiency in the cellulose hydrolysis

Siqueira et al. Biotechnol Biofuels 4:7, 2011 0 10 20 30 40 50 60 70 0 24 48 72 C e ll u lo s e c o n v e rs io n (% ) Hydrolysis time (h) 4h-treated (7 % of lignin) 2h-treated (12 % of lignin) 1h-treated (17 % of lignin) untreated (19 % of lignin)

Rind

0 10 20 30 40 50 60 70 0 24 48 72 Hydrolysis time (h) 2h-treated (7 % of lignin) 1h-treated (8 % of lignin) untreated (12 % of lignin)

Pith

Enzymatic hydrolysis: 10 FPU/g; 20 IU -glucosidase/g; @ 45 oC

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lignin contents

and

UV-data

in the

rind

Siqueira et al. Biotechnol Biofuels 4:7, 2011

R² = 0.940 R² = 0.927 R² = 0.655 0 10 20 30 40 50 60 70 0 0.1 0.2 0.3 0.4 0.5 C e llu lo s e c o n v e rs io n a ft e r 4 8 h ( % )

Absorbance of rind cells at 280 nm

vessel fiber parenchyma b R² = 0.973 R² = 0.959 R² = 0.969 0 10 20 30 40 50 60 70 0 0.2 0.4 0.6 C e llu lo s e c o n v e rs io n a ft e r 4 8 h ( % )

Absorbance of rind cells at 315 nm vessel fiber parenchyma c R² = 0.830 0 10 20 30 40 50 60 70 0 5 10 15 20 25 C e llu lo s e c o n v e rs io n a ft e r 4 8 h ( % )

Total lignin content in rind (%)

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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?

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What is different in the evaluated sugar cane regions?

Frequency of vascular bundles in varied fractions of a sugar cane internode

Volumetric and mass proportions of each sugar cane region in a sugar cane internode

Rind

Interface

Pith

Epidermis

Ep Rind Interface

Pith

Sugar cane region Number of vascular bundles/16mm2

Pith 6  1

Interface 8  1

Rind 13  2

Epidermis Only fibers

Sugar cane region

Volumetric proportion (%)

Dry mass proportion (%)

Pith 10 4.7  0.7

Interface 29 12  2

Rind 49 47  1

Epidermis 12 37  1

a medula representa menos do que 5% da massa total

Recordando

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Lee et al.., Biotechnol Bioeng, 2009 Selectively delignified Maple using ionic liquids

Siqueira et al., Applied Energy , 2013 Chlorite delignified sugarcane bagasse

Diminuindo a recalcitrância

um pré-tratamento é necessário

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0 5 10 15 20 25 30 35 0 24 48 72 C e ll u lo se co n ve rsi o n ( % ) Hydrolysis time (h) 89 146 87 8 53 121 50 58 166 321 mill bagasse 140 reference cultivar a R² = 0.749 0 5 10 15 20 25 30 35 15 17 19 21 23 25 27 C e ll u lo se co n ve rsi o n a fe tr 7 2 h o f e n zym a ti c h yd ro lysi s (% )

Total lignin content (%)

Lignin removal is necessary

to reach hydrolysis levels

over 70%

Direct enzymatic hydrolysis of milled sugar cane hybrids

y = -6.438x y = -8.011x 0 20 40 60 80 100 8 10 12 14 16 18 20 22 24 26 C e ll u lo se co n ve rsi o n a fe tr 7 2 h o f e n zym a ti c h yd ro lysi s (% )

Total lignin content (%)

chlorite-treated reference cultivar 24.5% Intial lignin

chlorite-treated hybrid 146

(18.6% initial lignin)

untreated hybrids

Masarin et al. Biotechnol Biofuels 4:55, 2011

- Plantas com menos lignina

são a solução??

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Chen and Dixon, Nature Biotechnology, 2007

Transgenic alfalfa

O total de polissacarídeos

hidrolisados deveria

atingir cerca de 900 mg/g

CWR

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- Remoção de lignina por processos industriais de pré-tratamento

são a solução??

hemicellulose

Lignin

Sulfito alcalino como agente deslignificante de bagaço de cana

Função dos componentes não celulósicos na recalcitrância da parede celular