Ridesa
Universidade Federal de Viçosa
Plant Biology Department
Plant Physiology Graduate Program
Molecular Plant Physiology Laboratory
Bioen Workshop on Metabolomics of Sugarcane
Monolignol profiling as selection tool for higher efficiency
of 2nd generation ethanol production from sugarcane bagasse
Marcelo Ehlers Loureiro
[email protected]
Marcio Henrique Barbosa
[email protected]
RIDESA
Rede Interuniversitária para o Desenvolvimento do Setor Sucroalcooleiro
(Academic Network for the Development of Sugar-Alcohol Sector)
Network responsable for
genotypes cultivated in 57% of
total sugarcane cultivated area
Successful interaction with private companies: no need of public money
High cost enzymes  major factor in cost
Lignin:
1) do not allow enzyme to attach to celullose
2) Trap the enzyme
3) Products of hydrolysis inhibit
enzyme and fermentation
Pectic matrix
Methylglucuronyl
Cellulose microfibril
arabinosyl
Nucleation site for
lignin deposition ?
GAX – (Glucurono)arabinoxylan
Gramine CW model
Gibeaut and Carpita 1993
Phenolics sheathing a
cellulose microfibrill
Lignin Composition Controlled by Genetic Manipulation and Monitored via Histochemical
Staining for Lignin Monomer Composition in Arabidopsis Stem Cross Sections.
Several evidences: lignin composition alteration do not
have dramatic effects as alteration in lignin content
Chapple et al 1988
(US-DOE 2006)
Evaluated Traits in some wild and domesticated sugarcane
Trait
S. Officinarum
(Domesticated)
S. Spontaneum
(wild)
S. Robustum
(wild)
RB867515
(cultivated
hybrid)
Sugar
Fibre
Yield
Ratooning
Pest
resistance
Exploitation of natural variation of sugarcane germplasms
High
medium
low
New directions to breeding programs
Conversion Efficiency
1st Generation
1) 86 L ethanol + 10.8 kg bagasse (DM) = 2200 MJ  efficiency = 29.8%
2) 86 L ethanol + 60 kWh = 2230 MJ  efficiency = 30.1%
Alcohol production using only sucrose : wasting of energy
Alcohol production using sucrose and burning bagasse for thermical energy :
still waste of energy: 70% of energy present in sugarcane is lost
62%
38%
59%
62%
Source: Rhao S. (2006)
Production of lignocellulosic alcohol
Possible to double the ethanol production:
increase from 6.000 L/ha to 12.000 L/ha
Potential : 1 ton cana→ 160 L cellulosic ethanol: 19000L/ha
Inverse correlation between sugar and fiber?
Average of three harvest experiments with 241 genotypes: 5800 determinations
New breeding strategies needed for secong generation biofuels for sugarcane
Genetic studies about heridity of lignin and fiber content at UFV
Variable Components of lignin content heredity (280 genotypes)
(Individual REML)
vg: Genetic variability,
vbloc: Variability in the units,
ve: External variability
vf: Phenotypic variability
h2g: Genotypic heritability
c2: Coefficient of interaction between experimental units
h2mgen : Heritability of Genotypic Average
vg
vbloc
ve
h2 g
0.897448
0.042468
1.519419
0.364915 +- 0.0927
vf
c2bloc
h2mgen
Média geral
2.459335
0.017268
0.364915
22.622275
Characterization of culm fiber content variability
20,00
18,00
14,00
12,00
10,00
8,00
6,00
Experiment in course about genetic of fiber content
4,00
2,00
5 higher and lower fiber content
Cultivation in three diferent environments
Analysis under course
0,00
2
3
4
6
9
12
13
14
15
16
17
19
20
22
23
26
27
31
32
35
37
38
39
41
44
45
46
50
52
53
54
55
62
65
66
67
69
71
72
73
77
78
79
80
83
87
91
93
94
96
97
98
99
10
content
fiber
Culm
de lignina (%)
média
Porcentagem
16,00
Genótipos de cana-de-açúcar
Sugarcane
genotypes
What does analytical pyrolysis means
Stepwise degradation of chemical bonds in organic compounds by gradative
increase of temperature in the absence of O2
Determination of best conditions
- Temperature
- Column
- Split of carrier gas : sample volatiles
- etc..
Pyrolysis in a quartz chamber
Volatilization and injection into GC column
Sample separation
Compound detection by an ionizing flame in the MS
Data collecting and generation (TIC)
Methods for large scale cell wall phenotype characterization
Pyrolysis+GC/MS: purified lignin from sugarcane stalks (TIC)
Pyrolysis+GC/MS: extracted cell walls from sugarcane stalks (TIC)
Similar values relative to
the results from purified lignin
Analytical time used:
1) Method with lignin purification
Purify lignin: 4 days
Pyrolysis + GC/MS = 60 min
2) Method without lignin purification:
Extraction: 1 hour
Pyrolysis + GC/MS = 60 min
A
B
Peak
Marker
Lignin type
8
Phenol
H
13
Guaiacol
G
18
4-Ethylphenol
G
20
4-Vinylphenol
H
28
Syringol
S
37
4-Methylsiringol
S
56
trans-4-(Prop-2-enyl) syringol
S
Nitrobenzene Oxidation
Pyrogram of Klason lignin (A) and bagasse
(B) of SP80-3280.
RPearson = 0,9857
Py-GC/MS
UFV: quick measurement 16 molignols, and S/G ratio
A
Sugarcane
Leaf cell wall
B
Arabidopsis
Crude root
Sample
Dirigent Mutant
Sugar cane culm
Arabidopsis roots
UFV:
Clones with lower lignin content
(2008)
aLignina Klason
(%)
S/G Ratio
146
22,6371
1,38
133
22,6494
1,23
58
22,6774
1,3
166
22,6782
53
22,7169
1,25
87
23,8285
1,08
8
24,0813
1,31
349
24,1626
1,36
321
24,4831
1,38
50
24,7027
1,22
∆ =8%
1,32
Clones with higher lignin content
(2008)
Populus Davison et al., 2006 :
∆ lignin = 12%
∆ S/G ratio = 21,7%
∆= 21,74%
Sharp increase in the acid
Hydrolysis
30%to 55%
Parents of actual hybrids under cultivation
Wild cane – S. spontaneum
S. officinarum
Spatial and temporal changes in lignin content
Spatial and temporal changes in lignin content
RB 867515
S. spontaneum
Spatial and temporal changes in lignin composition (Maule)
Spatial and temporal changes in lignin composition
RB 867515
S. spontaneum
S/G/H units in cell wall residue of internodes 1, 2 and 5
S
G
H
S/G
S. spontaneum
7,21
21,57
15,23
0,33
S. robustum
5,27
18,01
24,19
0,29
S. sinensis
3,82
16,12
15,46
0,23
RB867515
3,90
15,20
18,99
0,25
S/G/H units in sugarcane bagasse
S
G
H
S/G
RB867515
24,24
19,08
55,43
1,27
RB835486
31,085
25,95
42,275
1,19
SP1011
28,665
25,51
45,675
1,12
SP3280
28,475
27,19
42,91
1,04
SP1816
25,23
24,145
49,21
1,04
Characterization of polysaccharide structure by MALDI -TOF /MS
Allows for detection of minor structural changes in hemicellulose
- glicosylations
- Matrix type
Determination
of best conditions - Suitable enzymes
- acetylations
- methylations
- Ion mode, frame range selector , etc..
- arabinosylations, etc...
Cell wall extraction/components fractionation
Enzymatic Digestion
Sample/matrix co-crystallization
Laser Shots and collect data
Data analysis
Francis Lopes
PhD Thesis
Voyager Spec #1=>BC=>AdvBC(32,0.5,0.1)[BP = 377.3, 43269]
715.1516
100
Tree Genomics 2010
9.8E+3
90
missing
70
723.1280
60
% Intensity
MUR3 mutant
80
1247.2623
731.1132
missing
714.1471
50
1085.3064
706.1566
722.1288
40
737.1118
729.1420
745.1092
30
1248.2587
738.1013
732.1105
20
1086.3148
1263.2203
885.0660
728.1387
1101.2638
1249.2636
851.4203
10
0
700
900
1100
1300
0
1700
1500
Mass (m /z)
Voyager Spec #1=>AdvBC(32,0.5,0.1)[BP = 377.3, 35078]
1435.3430
1.0E+4
90
1393.3671
80
70
1436.3426
1394.3376
60
1555.3136
% Intensity
MUR 3 Mutant
complemented with an E.
grandis MUR3 gene
100
50
1247.3997
715.2262
1409.3024
1597.2841
1085.4311
40
1556.2858
1437.3152
30
714.2151
706.2305
1248.4113
0
1452.3116
791.4610
20 701.2256
10
1598.3042
1410.2998
707.2471
745.1974
730.1831
761.7061
709.6892
763.6778
953.4667
851.4874
1013.4369
815.9027
829.8804
907.0635
1453.2978
1263.3164
1086.3882
1411.3124
1175.3703
1101.3257
1115.4467
1599.2629
1613.2177
1572.2238
1264.3893
1305.3069
1600.3301
0
Xyloglucan structure phenotyping in A. thaliana by MALDI TOF MS
analysis
Nomenclature according to Fry et al., 1993
Glucose (G)
Xylose (X)
Galactose (L)
Fucose (F)
Intens. [a.u.]
MALDI-TOF MS spectra of oligossacharides from xylanase digestion of internodes 1, 2 and 5
637.265
G13 _1tec2 Man xil\0_G13\1\1SRef
Saccharum sinensis
919.448
6000
1091.665 1265.844
4000
1397.972
1001.542
785.279
1572.110
2000
1746.237
1920.336
Intens. [a.u.]
0
x104
G15 _1 IM76 xil TecRep2\0_G15\1\1SRef
TecRep2\0_G15\1\1SRef
Saccharum
robustum
1.25
1.00
637.339
0.75
0.50
833.420
1013.661 1175.855 1338.000
1500.168
0.25
Intens.
[a.u.]
Intens. [a.u.]
0.00
G18 _2 IN84 Xil TecRep1\0_G18\1\1SRef
TecRep1\0_G18\1\1SRef
571.833
Saccharum spontaneum
6000
4000
607.381
2000
2000
2397.838
2397.838
764.715
764.715
Intens. [a.u.]
0
0
x104 572.784
600
1.0
800
1497.261
1497.261
1000
1200
1400
1600
G19 _1 RB7515
1800
2000 xil TecRep2\0_G19\1\1SRef
2200
2400
RB867515m/z
611.049
919.440
0.8
1093.646
0.6
637.268
0.4
785.270
1265.820
1001.540
1439.971
0.2
1614.093
1788.191
0.0
600
800
1000
1200
1400
1600
1800
2000
2200
2400
Intens. [a.u.]
Intens. [a.u.]
S. spontaneum cell wall digestion of resistant residue (internodes 1, 2 and 5)
Xylanase (8mM oxalic acid)
Xylanase (32 mM oxalic acid)
550.379
1376.649
4000
522.304
4000
851.224
689.150
1013.280
3000
3000
554.487
1175.334
2000
2000
1337.378
851.356
1013.568
1661.425
689.123
1175.751
1499.425
1337.909
1000
1261.624
1000
615.356
615.259
579.486
644.601
1064.783
590.515
1300.539
1276.959
1376.266
735.224
913.505
0
0
500
600
700
800
900
1000
1100
1200
1300
600
1400
800
1000
1200
1400
1600
m/z
m/z
Driselase (32mM oxalic acid)
Intens. [a.u.]
Intens. [a.u.]
Driselase (8mM oxalic acid)
x104
522.384
x104
2.5
579.311
3
550.442
2.0
2
1.5
851.350
1013.550
1175.734
689.126
881.405
1.0
1337.894
607.484
1
1500.028
1662.137
0.5
637.322
1824.244
579.412
607.367
1175.825
1986.331
1091.653
1337.977 1393.308
1572.046
2148.384
2310.433
0
500
600
700
800
900
1000
1100
1200
1300
1400
m/z
2472.450
0.0
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
m/z
Partial Oligossacharide profiling of RB867515 with xylanase
Relative intensity
X2GlcA1OAc1
X4OAc1
1,2
X3GlcA (1FerA)1OAc1
X5
X5OAc1
1
X5OAc2
X4GlcA(FerA)1OAc1
X4GlcA(FerA)1OAc2
0,8
X6
X5G
X6OAc2
0,6
X5GlcA1OAc1
X5GlcA(FerA)1OAc2
X7
X7OAc2
0,4
X6GlcA(FerA)1OAc1
X6GlcA(FerA)1OAc2
X8
0,2
X7GlcA(FerA)1OAc1
X7GlcA(FerA)1OAc2
X9
0
1
Cell wall material of internodes 1,2 and 5
X8GlcA(FerA)1OAc2
X10
Functional genomics of sugarcane cell wall
Full-length libraries builded yet: internode 1-2 and 4-5 sugarcane (867515)
-Library transformation of 23 cell homozigote cell wall mutants of Arabidopsis
and phenotypic evaluation.
- Pirosequence and annotation (Glaucia USP)
Our basic infraestructure:
 5 greenhouses (1 for transgenics), and almost all equipment needed for basic
Physiological characterizations: IRGAs (3), fluorometers (2), image fluorescence
High resolution infrared camera, Scholander pump, osmometer,etc
-Very good analytical equipment for metabolomics and proteomics
-Two tissue culture labs (Marcio and Marcelo), and a molecular phisiology lab (real
time PCR, nanoHPLC, elisa readers...)
Sugarcane group – Molecular Plant Physiology lab/UFV
Valdir Diola-Post Doc (Full-lenght,Plant transformation), David Baffa-MS student (Genetic breeding
Viviane-PhD student (transcriptomics), Francis Lopes-Post Doc (Metabolomics)