RIND - Departamento de Biotecnologia

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 sugarcane cell walls and its correlation with the enzymatic hydrolysis of
polysaccharides. Biotechnol Biofuels 4:1–9
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)
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 models
Cell wall SEM
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
Glicose (g/L)
Glicose (g/L)
 2% Acid
Glicose (g/L)
Xilose (g/L)
Xilose (g/L)
Xilose (g/L)
Máx. esperados: Xilose = 26 g/L
 2% Acid
 2% Acid
 4% Acid
▲ 6% Acid
 4% Acid
▲ 6% Acid
 4% Acid
▲ 6% Acid
Glicose = 42 g/L
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. 46:1801-1809, 2006.
Gramíneas como modelo de estudos
devido a grande variabilidade de tipos
celulares
Cell anatomy in sugar cane
vascular bundles
Rind
Pith
Enzymatic hydrolysis of varied sugar cane regions from the internode
Epidermis
Rind
~ 3 cm
Interface
Pith
Rind
100
Cellulose conversion (%)
Epidermis
80
Pith
Interface
Rind
Epidermis
60
40
Interface
20
Pith
0
0
20
40
Hydrolysis time (h)
60
80
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
Epidermis
Sugar cane region
Pith
Interface
Rind
Epidermis
Number of vascular
bundles/16mm2
61
81
13  2
Only fibers
Rind
Interface
Pith
Volumetric and mass proportions of each sugar
cane region in a sugar cane internode
Ep Rind Interface Pith
Sugar cane
region
Pith
Interface
Rind
Epidermis
Volumetric
proportion (%)
10
29
49
12
Dry mass proportion
(%)
4.7  0.7
12  2
47  1
37  1
Chemical composition of varied fractions in a sugar cane internode
Sugar
cane
region
Pith
Interface
Rind
Epidermis
Chemical composition (%, w/w)
Extractives
Glucan
Xylan
Arabinosyl
Acetyl
Insoluble
lignin
Soluble
lignin
2.9  0.2
3.0  0.1
1.3  0.1
3.1  0.1
49.3  0.7
43.8  0.1
42.0  0.5
42.4  0.5
15.4  0.2
18.8  0.1
20.4  0.2
19.6  0.2
0.1  0.1
1.4  0.1
1.1  0.1
1.2  0.1
2.4 0.2
3.2  0.1
3.0  0.2
2.6  0.2
14.8  0.1
20.4  0.1
19.8  0.1
20.4  0.1
2.4 0.1
2.4  0.3
2.0  0.3
1.9  0.2
Hydroxycinnamic acids (%, g/100g of in natura bagasse)
Pith
Interface
Rind
Epidermis
Released by mild-alkaline treatment
Ferulic
Coumaric
Sum
0.49  0.05
1.4  0.2
1.9  0.2
0.50  0.08
2.0  0.4
2.5  0.4
0.48  0.06
2.1  0.2
2.7  0.1
0.34  0.08
1.3  0.4
1.6  0.5
Released by severe-alkaline treatment
Ferulic
Coumaric
Sum
1.5  0.1
5.3  0.7
6.8  0.7
1.6 0.1
8.1  0.8
9.7  0.9
1.4  0.1
6.7  0.4
8.1  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
Example of a selected point (1 µm2) to
record the UV spectrum
UV-microscope
400 x
Average spectrum from different cells in sugar cane
Rind
Pith
0.6
Absorbance
0.4
vessel
fiber
parenchyma
0.3
0.2
0.5
Absorbance
0.5
0.6
0.4
0.3
0.2
0.1
0.1
0
0
-0.1
220 240 260 280 300 320 340 360 380 400
Wavelenght (nm)
vessel
fiber
parenchyma
-0.1
220 240 260 280 300 320 340 360 380 400
Wavelenght (nm)
280 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, 2011
Frequency histograms
0.3
0.2

Weighed-average Absorbance
0.1
0
0
0.12
0.20
0.26
0.33
0.39
0.46
0.52
0.59
0.65
0.72
0.78
0.85
0.91
0.97
Frequence of pixels
Topochemical
distribution of lignin
Absorbance values range
Rind fibers
Abs280 nm=0.40
Pith fibers
Abs280nm=0.39
Similar lignin contents
Rind Parenchyma
Abs280nm = 0.31
Pith Parenchyma
Abs280nm = 0.18
Lower lignin content in
pith parenchyma
Selective delignification of rind and pith regions
RIND
0.5
vessel
a
0.6
untreated
1-h treated
2-h treated
Absorbance
0.4
0.5
0.3
0.2
0.1
0.2
0.1
0
-0.1
-0.1
220 240 260 280 300 320 340 360 380 400
Absorbance
b
fiber
220 240 260 280 300 320 340 360 380 400
Wavelenght (nm)
0.6
0.5
untreated
1h-treated
2h-treated
0.4
0.3
0.2
0.1
0.2
0.1
0
-0.1
-0.1
220 240 260 280 300 320 340 360 380 400
220 240 260 280 300 320 340 360 380 400
0.6
0.6
Wavelenght (nm)
0.3
untreated
1-h treated
2-h treated
0.2
0.5
parenchyma
untreated
0.3
1-h treated
0.2
0.1
0.1
0
0
-0.1
Wavelenght (nm)
0.4
Absorbance
parenchyma
c
0.4
Absrobance
untreated
1-h treated
0.3
0
0.5
Wavelenght (nm)
fiber
0.4
Absorbance
0.6
0.5
untreated
1-h treated
0.3
0
UV-data for
rind and pith
regions after
acetic
acid/chlorite
delignification
a
vessel
0.4
Absorbance
0.6
PITH
-0.1
220 240 260 280 300 320 340 360 380 400
Wavelenght (nm)
220 240 260 280 300 320 340 360 380 400
Wavelenght (nm)
Cell type
Lignin removal
from recalcitrant
rind regions
UV-images from
sugar cane
internode cells
>> after chloriteinduced
delignification
Siqueira et al.
Biotechnol Biofuels 4:7, 2011
Untreated
fibers
1-h chlorite/
acetic acid
treated fibers
Untreated
parenchyma
1-h
acetic
acid/chlorite
treated
parenchyma
Rind
Cellulose conversion (%)
Lignin removal (chlorite delignification) versus
efficiency in the cellulose hydrolysis
70
70
Rind
60
Pith
60
50
50
40
4h-treated (7 % of lignin)
2h-treated (12 % of lignin)
1h-treated (17 % of lignin)
untreated (19 % of lignin)
30
20
40
2h-treated (7 % of lignin)
30
1h-treated (8 % of lignin)
20
untreated (12 % of lignin)
10
10
0
0
0
24
48
Hydrolysis time (h)
Enzymatic hydrolysis: 10 FPU/g;
20 IU -glucosidase/g; @ 45 oC
72
0
24
48
72
Hydrolysis time (h)
Siqueira et al. Biotechnol Biofuels 4:7, 2011
Cellulose conversion after
48 h (%)
Correlation of enzymatic hydrolysis efficiency with:
lignin contents
and
UV-data
in the rind
70
60
50
40
30
20
R² = 0.830
10
0
0
5
10
15
20
25
Cellulose conversion after
48 h (%)
70
b
vessel
fiber
parenchyma
60
50
40
30
20
10
R² = 0.655
R² = 0.927 R² = 0.940
0
0
0.1
0.2
0.3
0.4
0.5
Absorbance of rind cells at 280 nm
Siqueira et al. Biotechnol Biofuels 4:7, 2011
Cellulose conversion after
48h (%)
Total lignin content in rind (%)
70
vessel
fiber
parenchyma
c
60
50
40
30
20
10
R² = 0.969
R² = 0.959
R² = 0.973
0
0
0.2
0.4
Absorbance of rind cells at 315 nm
0.6
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?
Recordando
What is different in the evaluated sugar cane regions?
Frequency of vascular bundles in varied fractions of a sugar cane internode
Epidermis
Sugar cane region
Pith
Interface
Rind
Epidermis
Number of vascular
bundles/16mm2
61
81
13  2
Only fibers
Rind
Interface
Pith
Volumetric and mass proportions of each sugar
cane region in a sugar cane internode
Ep Rind Interface Pith
Sugar cane
region
Pith
Interface
Rind
Epidermis
Volumetric
proportion (%)
10
29
49
12
Dry mass proportion
(%)
4.7  0.7
12  2
47  1
37  1
a medula representa menos do que 5% da massa total
Diminuindo a recalcitrância
um pré-tratamento é necessário
- Quanta lignina deveria ser removida?
Lee et al.., Biotechnol Bioeng, 2009
Selectively delignified Maple using
ionic liquids
Siqueira et al., Applied Energy , 2013
Chlorite delignified sugarcane bagasse
- Plantas com menos lignina
são a solução??
35
89
Cellulose conversion (%)
a
30
146
25
8
87
53
121
20
50
58
15
Cellulose conversion afetr 72h of
enzymatic hydrolysis (%)
Direct enzymatic hydrolysis of milled sugar cane hybrids
35
30
25
20
R² = 0.749
15
10
5
0
15
166
17
19
21
23
25
27
321
10
mill bagasse
5
Total lignin content (%)
140
0
24
48
72
Hydrolysis time (h)
Lignin removal is necessary
to reach hydrolysis levels
over 70%
Cellulose conversion afetr 72h of
enzymatic hydrolysis (%)
reference cultivar
0
100
chlorite-treated reference cultivar
24.5% Intial lignin
80
y = -6.438x
60
chlorite-treated
hybrid 146
(18.6% initial lignin)
40
y = -8.011x
20
untreated hybrids
0
8
Masarin et al. Biotechnol Biofuels 4:55, 2011
10
12
14
16
18
20
22
Total lignin content (%)
24
26
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??
Sulfito alcalino como agente deslignificante de bagaço de cana
hemicellulose
Lignin
Mendes et al. Progress Biotechnol, 27:395-401, 2011