1. Effect of Different Pretreatment Methods in Combination
with the Organosolv Delignification Process and
Enzymatic Hydrolysability of Three Feedstocks in
Correlation with Lignin Structure
Yakindra Prasad Timilsena
Examination Committee
 Prof. Sudip K. Rakshit (Chairperson)
 Prof. Nicolas Brosse (Co-advisor)
 Prof. Athapol Noomhorm
 Dr. Anil Kumar Anal

2. Overview of Presentation
1 Introduction
2 Review of Literatures
3 Materials and Methods
4 Results and Discussions
5 Conclusions
2

3. Introduction
Lignocellulosics
→ Plentiful supply & Renewable resources
→ Comparatively low cost
→ No competition with food and feed production
→ Environmentally benign
3

4. Problem Statement
Introduction Background
Objectives
Potential sources
• Dedicated crops
• Invasive plants
• Agro-industrial waste
4

5. Problem statement
Introduction Background
Objectives
Lignocellulosic Conversion Process
• Direct combustion
• Biogas (Gasification, Anaerobic digestion)
• Biofuels (Bioethanol, biomethanol, Fischer-
Tropsch (FT) diesel, biobutanol,
Biohydrogen)
Conversion to
Holocellulose monomeric sugars
Lignocellulosics
Lignin Conversion to
renewable
Adhesive, biodegradable fuel, chemicals or
polymers, bioantioxidant,
food
5

6. Lignocellulosics Lignocellulosics Application
6

7. Pretreatment
Problem statement
Background
Objectives
 Important processing step in biomass
conversion
 alter the structure of the biomass
 break the lignin seal
 disrupt the crystalline structure of cellulose
(Adapted from Hsu et al., 1980).
7

8. Background
Introduction Objectives
Methods of pretreatment
Methods
Combinative pretreatment
8

9. Problem Statement
Problem statement Background
Objectives
• Selection of feedstock (Composition, growth
requirement, productivity, land/water
competition with food or fodder, biomass
nature and ease of delignication and pulp yield)
• Selection of pretreatment method (long list of
optimized methods, difficult to choose)
9

10. Problem Statement
Rationale Background
Objectives
• Molecular structure of constituent
polymers, especially lignin
10

11. Background
Objectives Objectives
Objective 1 Objective 2 Objective 3
To compare the
To characterize
and describe To evaluate the
delignification Typha lignin and effect of
ability of different establish aromatic
prehydrolysis correlation compounds in
methods and to
between lignin organosolv
structure (S/G delignification
assess the ratio) and ability of
effectiveness of delignification Miscanthus
pretreatment ability
11

12. Literature Review
S.N. Feed- Pretreatment Findings Author &Year
stock method
1. Aspen Autohydrolysis & - Positive effect of aromatics in Wayman &
solvent extraction delignification Lora, 1978
2. Bagasse Presoaking, - Better effect of prehydrolysis Patel &
prehydrolysis+ Varshney, 1989
organosolv
delignification
3. MxG DAP + Ethanol - Presoaking has better effect Brosse et al.,
organosolv process on xylan recovery, 2009
delignification and enzymatic
digestibility
4. MxG Lignin - Description of two kinds of El Hage et al.,
characterization lignin from MxG 2009
5. MxG Autohydrolysis + -Autohydrolysis enhanced the El Hage et al.,
OS delignification 2010
- Positive effect of 2-naphthol
12

13. MATERIALS & METHODS
Pressure
Guaze
Reactor
Heater
Temp.
Controller
13

14. Raw Materials
Raw Materials Prehydrolysis
Organosolv Process
Enzymatic Hydrolysis
Lignin Separation & Characterizati
Miscanthus x Giganteus
energy dedicated crop Palm oil industry
Perennial grass
agricultural by-product
Non invasive low cost
Requires no nitrogen / herbicide 6 million tons /year in Malaysia
Produces 20-25 tons /ha/year
Typha capensis
invasive grass
fast growing , highly
prolific (50-60 ton/ha/year)

15. OBJECTIVE 1
To compare the delignification ability of different
prehydrolysis methods and to assess the
effectiveness of pretreatment
• Pulp yield
• EOL yield
• KL content of the pulp
• Total reducing sugar and glucose yield
15

16. Raw Materials
Materials and Methods Prehydrolysis
Organosolv Process
Enzymatic Hydrolysis
Lignin Separation & Characterizati
16

17. Raw Materials
Materials and Methods Prehydrolysis
Organosolv Process
Enzymatic Hydrolysis
Second step: Organosolv Delignification Lignin Separation & Characterizati
17

18. Composition
Results and Discussions Prehydrolysis
Combinative pretreatment
Enzymatic hydrolysability
1. Composition of untreated biomass Lignin Characterization
100
Content (% extractive free dry wt basis)
90 23.1 20.4
25.9 Lignin (%)
80 Hemicellulose (%)
70 Cellulose (%)
60 26.7 28.5 38.4
50
40
30
47.4 48.4
20 41.2
10
0
MxG EFB Typha
Feedstock
• Holocellulose extraction by sulphite
 Glucans and xylans: 75-80% delignification method
• Cellulose extraction by alkaline method
 Lignin: 20-25% (TAPPI)
• Lignin by difference
 Composition almost similar for all biomasses
18

19. Composition
Composition of Prehydrolysis
untreated biomass
Combinative pretreatment
Enzymatic hydrolysability
Lignin Characterization
Composition after prehydrolysis
 Xylans hydrolysed and removed in large amount (Typha>EFB>MxG)
 Partial lignin removal
 Pulp rich in cellulose
19

20. Composition
Results and Discussions Prehydrolysis
Combinative pretreatment
Enzymatic hydrolysability
Lignin Characterization
 Lignin substantially removed
 Higher mass loss
20

21. Composition
Results and Discussions Prehydrolysis
Combinative pretreatment
Enzymatic hydrolysability
EOL & KL Content of the pulp after
Lignin Characterization
organosolv delignification
EOL
18
KL
Percentage (dry biomass basis)
20.0 17
16 16
18.0 16
14
16.0 13 12
14.0 12 12 11
12 10
12.0
8
10.0 8 8 8
7 1 6 7
8.0 6 5 6 6
5 5 5 5 6 6
6.0
4.0 2
2.0 1 1
0.0
Miscanthus EFB Typha
Treatments
 Prehydrolysis step enhanced the subsequent delignification (destruction of lignin
seal, easier delignification)
 DAP, SP & EP: not very efficient (significant delignification of EFB in DAP)
 Naphthol : positive effect for MxG and EFB; no effect in Typha
 Typha has different behaviour; easier to delignify even with single step pretreatment. 21

22. Composition
Results and Discussions Prehydrolysis
Combinative pretreatment
MxG: Yield of total reducing sugars and glucose Enzymatic hydrolysability
Lignin Characterization
70
61
Reducing sugar 58
60
Glucose
52
50 49
50
45
44
Sugar content (%)
39
40 38
33
29 30
30 28
25
24
20
15
11
10 6
7
3
0
RM_M DAP AHN SP OS DAP+OS AH + OS EP + OS AHN + OS SP+OS
Treatments
 low hydrolysability after prehydrolysis
 low hydrolysability after organosolv alone (performed at low severity, low conc. of sulfuric
acid, low temperature..)
 hydrolysability enhanced after combinative treatment. Organosolv is necessary because it
removes a large part of lignin and make cellulose more accessible 22

23. Composition
Results and Discussions Prehydrolysis
Combinative pretreatment
EFB: Yield of total reducing sugars and glucose Enzymatic hydrolysability
Lignin Characterization
70
64
Reducing sugar 61
60
Glucose 54
50
49
50
44
Sugar content (%)
39
40
34
33 32 32
29
30 27 26
24
22 22
19
20
10
10
5
0
RM_E DAP AHN SP OS DAP+OS AH + OS EP + OS AHN + OS SP+OS
Treatments
 good correlation was observed between Lignin content & hydrolysability
 Dilute acid prehydrolysis+Organosolv process showed best result.
23

24. Composition
Results and Discussions Prehydrolysis
Combinative pretreatment
Typha: Yield of total reducing sugars and glucose Enzymatic hydrolysability
Lignin Characterization
70
Reducing sugar 60 61
59
60 Glucose
57 58
53
50 46
45 43
42 43 43
41
Sugar content (%)
39 39
40 36
35 34 34
29
30 27
21
20
13
10 6
0
 Typha demonstrated different behaviour
Treatments
 Good hydrolysability after the prehydrolysis even if the KL content in the pulp are high
 Reactivity toward enzyme only slightly improved after OS
 Typha is easier to delignify, one step process showed tantamount effect
 No effect of naphthol on delignification ability
24

25. OBJECTIVE 2
To characterize and describe Typha lignin and
establish correlation between lignin structure (S/G
ratio) and delignification ability
• FTIR • Major peak assignment &
• NMR description
• GPC • Relative amount of constituent
moieties (S/G ratio)
• Mn, Mw & PI
25

26. Raw Materials
Materials and Methods Prehydrolysis
Organosolv Process
Enzymatic Hydrolysis
Lignin Separation & Characterizati
Lignin Isolation
26

27. Raw Materials
Materials and Methods Prehydrolysis
Organosolv Process
Enzymatic Hydrolysis
Lignin Separation & Characterizati
Lignin characterization
• Two fractions of lignin analysed (CEL & EOL)
• Spectroscopic methods (FTIR & NMR)
• Chromatographic method (GPC)
27

28. Composition
Results and Discussions Prehydrolysis
Combinative pretreatment
Enzymatic hydrolysability
Lignin Characterization
Lignin polymer
• Lignin is a complex natural polymer
comprised of p-hydroxyphenyl (H),
guaiacyl (G) and syringyl (S) units
• (S/G) ratio- important characteristic
(because G has high tendency to
recondensed >> delignification more
difficult)
H G S
Adapted from Wershaw, 2004
28

29. Composition
Results and Discussions Prehydrolysis
Combinative pretreatment
FTIR spectra of Typha CEL Enzymatic hydrolysability
Lignin Characterization
70,5
65
- 1515.9 G+S
- 1329.5 S
60 2065,0
- 1240 G
55
3854,3
- 1166.8 typical of
50 3821,4 HGS lignin
- 1125.9S
45 2341,5
- 1033.8 G
2360,0
40
- 834.7 G+S
35
%
T
30
25 1166.8
20 834,7
668,3
15 1369,4 605,6
527,5
10 1125,9
2933,8 1240,1
1729,9
5 3412,1
1329,5 1033,8
1515,9
1604,6 1457,3
3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400,0
Wavelength (cm-1)
 H-G-S lignin (usual for herbaceous crop)
29

30. Composition
Results and Discussions Prehydrolysis
Combinative pretreatment
FTIR Peak assignment for Typha CEL Enzymatic hydrolysability
Lignin Characterization
- 1515.9 G+S
- 1329.5 S
- 1240 G
- 1166.8 typical of HGS lignin
- 1125.9S
- 1033.8 G
- 834.7 G+S
30

31. Composition
Results and Discussions Prehydrolysis
Combinative pretreatment
NMR spectra of Typha CEL Enzymatic hydrolysability
Lignin Characterization
 Presence of residual sugars (peaks at 95-100ppm & 70-75ppm)
 High Acetylation content: 0.44 acetate group/aryl (0.06 for Miscanthus)
 Low paracoumaryl content : 0.01 PC group/aryl (0.1 for MxG)
 S/G/H= 55/15/30
 Very high S/G ratio (3.7)
 High S/G ratio support the easier delignification (Del Río et al., 2005)
31

32. Composition
Results and Discussions Prehydrolysis
Combinative pretreatment
NMR spectra of Typha CEL (A) & EOL (B) Enzymatic hydrolysability
Lignin Characterization
Comparison Typha EOL and CEL
 EOL non sugar (peaks at 95-
100ppm + 70-75ppm)
 propyl side chain shows
deconstruction of -O-4 linkage
(60-90ppm)
 Acetate extensively
153 147
hydrolysed during organosolv
hydrolysis
 In EOL, S etherified (153ppm
is very low but S non etherified
(147ppm) very high
extensive depolymerization
through aryl ether bond
cleavage. 32

33. Comparison of lignin from three feedstocks
33

34. Composition
Results and Discussions Prehydrolysis
Combinative pretreatment
SEC analysis of Typha CEL and EOL Enzymatic hydrolysability
Lignin Characterization
Lignin Mw Mn PI=Mw/Mn
EOL 4567 2877 1.59
CEL 9268 4109 2.26
• Higher molecular weight and polydispersity index of CEL
• Cleavage of aryl ether bonds & formation of smaller fragments during organosolv process
• Agreed with NMR results
34

35. Composition
Results and Discussions Prehydrolysis
Combinative pretreatment
SEC analysis of Typha CEL and EOL Enzymatic hydrolysability
Lignin Characterization
Lignin Mw Mn PI=Mw/Mn
CEL 9268 4109 2.26
• Higher molecular weight and polydispersity index of CEL
• Cleavage of aryl ether bonds & formation of smaller fragments during organosolv process
• Agreed with NMR results
35

36. OBJECTIVE 3
To evaluate the effect of aromatic compounds in
organosolv delignification ability of Miscanthus
2-naphthol
• p- cresol
• o-cresol
• EOL yield
• Klason lignin
• hydroquinone
content of the pulp
• Acid soluble lignin
• dihydroxyanthraquinone
36

37. Raw Materials
Materials and Methods Prehydrolysis
Organosolv Process
Enzymatic Hydrolysis
Lignin Separation & Characterizati
10 g ODW Miscanthus
Mixed with 0.4 g aromatics and soaked in
100 mL acetone overnight
Acetone evaporation by air drying
Autohydrolysis (1500C, 8h, S/L=1:9)
OS delignification (1700C, 1h, SA=0.5%, S/L=1:8)
Filtration Liquid EOL
phase
KL Pulp
37

38. Effect of aromatics on delignification
30 EOL yield (% )
KL (%)
25
Yield (%)
20
15
10
5
13.3 14.9 20.8 5.9 16.8 7.3 23.4 3.8 17.8 8.3 22 4.8
0
Control Naphthol o-Cresol p-Cresol Hydroquinone DHAq
Treatments
38

39. Effect of aromatics on delignification
39

40. Scavenging action of aromatics
path 1 occurs if the blue
For feedstocks with the fragment is a G unit
low G content, path 0 is (more reactive). >>
favoured Path 0 important for MxG
40

41. Conclusions
• Despite a very similar chemical composition, three biomasses
demonstrated different behavior during pretreatment.
• Typha was easier to delignify; one step pretreatment (prehydrolysis or
delignification) process was sufficient to break the lignin seal and
release the sugars for enzymatic action. The combinative pretreatment
not necessary
• The first step of pretreatment (i. e. prehydrolysis) significantly enhance
the efficacy of the second step of delignification of MxG and EFB and
enzymatic hydrolysability also. DAP plus OS pretreatment resulted into
best results for EFB. Autohydrolysis in presence of naphthol plus OS
pretreatment (AHN) is best for MxG.
• The treatment of biomass with a catalytic amount of aromatic
compounds like 2-naphthol during autohydrolysis exhibited a
substantial effect on both MxG and EFB delignification as well as on
enzymatic hydrolysability.
41

42. Conclusions
• Typha lignin is of H-G-S nature as usual to other herbaceous plants but
with high S/G ratio suggesting its easier delignification
• Addition of catalytic amount of aromatic scavengers enhanced
delignification substantially (2-naphthol, p-cresol and
dihydroxyanthraquinone with tantamount effect)
• a better knowledge of biomass at the molecular level allow a better
optimization of pretreatment
42

43. Recommendations
• Obnoxious Typha an interesting guinea-pig for tropical
biorefinery sector. Additional research for its valorization
essential.
• Experimentation on effect of additional aromatic
scavengers in various feedstocks essential.
43

44. INDEBTED TO
SDCC / AIT –
France Network
44

45. « Think global, act local »
GREEN
PROCESS
Green Energy
Green World
THANKS FOR YOUR ATTENTION!!!