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

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

polysaccharides. Biotechnol Biofuels 4:1–9

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

Revendo alguns conceitos

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 hidrolisar celulose??

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

Máx. esperados: Xilose = 26 g/L Glicose = 42 g/L

Xilo

se (

g/L

) X

ilose

(g

/L)

Xilo

se (

g/L

)

Glic

ose

(g

/L)

Glic

ose

(g

/L)

Glic

ose

(g

/L)

2% Acid 4% Acid

▲ 6% Acid

2% Acid 4% Acid

▲ 6% Acid

2% Acid 4% Acid

▲ 6% Acid

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

Variação natural de recalcitrância em diferentes tipos celulares

Cell anatomy in sugar cane

Rind Pith

vascular bundles

Gramíneas como modelo de estudos devido a grande variabilidade de tipos celulares

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

Ce

llu

lose

co

nve

rsio

n (%

)

Hydrolysis time (h)

PithInterfaceRindEpidermis

Epidermis

Rind

Interface

Pith

~ 3 cm

Enzymatic hydrolysis: 10 FPU/g;

20 IU -glucosidase/g; @ 45 oC

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

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

Lignina como maior causador natural da recalcitrância? Há evidências experimentais?

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

400 x

Average spectrum from different cells in sugar cane

280 nm > aromatic ring substituted with oxygenated groups

315 nm > aromatic ring conjugated with alfa-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

Ab

so

rba

nce

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

Ab

so

rba

nce

Wavelenght (nm)

vesselfiberparenchyma

Rind Pith

Siqueira et al. Biotechnol Biofuels 4:7, 2011

Similar lignin contents

Rind fibers Abs280 nm=0.40

Pith fibers Abs280nm=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

0.6

5

0.7

2

0.7

8

0.8

5

0.9

1

0.9

7

Fre

quence o

f pix

els

Absorbance values range

Weighed-average Absorbance

Rind Parenchyma Abs280nm = 0.31

Lower lignin content in pith parenchyma

Pith Parenchyma Abs280nm = 0.18

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

Ab

so

rbance

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

Ab

so

rbance

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

Ab

sro

bance

Wavelenght (nm)

untreated

1-h treated

2-h treated

cparenchyma

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

Ab

so

rbance

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

Ab

so

rbance

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

Ab

so

rbance

Wavelenght (nm)

untreated

1-h treated

vessel

fiber

parenchyma

UV-data for

rind and pith

regions after

acetic

acid/chlorite

delignification

Selective delignification of rind and pith regions

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

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

Ce

llu

lose

co

nve

rsio

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

lignin contents

and

UV-data

in the rind

Siqueira et al. Biotechnol Biofuels 4:7, 2011

R² = 0.940R² = 0.927R² = 0.655

0

10

20

30

40

50

60

70

0 0.1 0.2 0.3 0.4 0.5

Ce

llulo

se

co

nve

rsio

n a

fte

r 4

8 h

(%

)

Absorbance of rind cells at 280 nm

vesselfiberparenchyma

b

R² = 0.973R² = 0.959R² = 0.969

0

10

20

30

40

50

60

70

0 0.2 0.4 0.6

Ce

llulo

se

co

nve

rsio

n a

fte

r 4

8h

(%

)

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

Ce

llulo

se

co

nve

rsio

n a

fte

r 4

8 h

(%

)

Total lignin content in rind (%)

Correlation of enzymatic hydrolysis efficiency with:

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?

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

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

- Quanta lignina deveria ser removida?

0

5

10

15

20

25

30

35

0 24 48 72

Ce

llu

lose

co

nve

rsio

n (%

)

Hydrolysis time (h)

89

146

87

8

53

121

50

58

166

321

mill bagasse

140

reference cultivar

aR² = 0.749

0

5

10

15

20

25

30

35

15 17 19 21 23 25 27

Ce

llu

lose

co

nve

rsio

n a

fetr

72

h o

f e

nzym

atic h

yd

roly

sis

(%

)

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

Ce

llu

lose

co

nve

rsio

n a

fetr

72

h o

f e

nzym

atic h

yd

roly

sis

(%

)

Total lignin content (%)

chlorite-treated reference cultivar24.5% Intial lignin

chlorite-treatedhybrid 146

(18.6% initial lignin)

untreated hybrids

Masarin et al. Biotechnol Biofuels 4:55, 2011

- Plantas com menos lignina são a solução??

Chen and Dixon, Nature Biotechnology, 2007

Transgenic alfalfa

O total de polissacarídeos hidrolisados deveria atingir cerca de 900 mg/g CWR

- Remoção de lignina por processos industriais de pré-tratamento são a solução??

hemicellulose

Lignin

Sulfito alcalino como agente deslignificante de bagaço de cana

Mendes et al. Progress Biotechnol, 27:395-401, 2011