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