1. Master Thesis
May 2010
New Pretreatment Methods for Lignocellulosic Residue for Second Generation Bioethanol Production
Student:
Yadhu Nath Guragain
ID:
Adviser
Prof. Sudip K. Rakshit

2. Agenda Background of Study Objectives Materials & Methods
Results & Discussion
Objectives
Conclusion & Recommendations

3. Background of Study Global Energy Scenario Possibility Hurdles
Demand gradually Increasing
Major source, fossil fuel, Depleting
Climate change (GHG emission) Alarming
Possibility
Substitution of gasoline by Bioethanol
Hurdles
1st generation (from sugar and starchy materials)
:Food security challenge
:Limited feedstock
2nd generation (from non-edible parts of plants)
: Commercial production not economically viable

4. Production Process of 2nd Generation Bioethanol
Hemicelluloses
Cellulose
Lignin
Monomer Sugars
1. Pretreatment
2. Hydrolysis
3. Fermentation
Lignocellulose
Ethanol (ca. 10%)
4. Distillation
A lot of challenges in each steps
Major bottleneck: - Pretreatment
Pure Ethanol (ca. 99.5%)

5. Objective of pretreatment
To break down lignin-hemicelluloses-cellulose complex, making carbohydrate polymers susceptible for enzymatic hydrolysis
Existing Situation:
Number of biological, physical and chemical methods available.
For e.g. enzyme, ball milling, steam explosion, acid, alkali and so on.
Yet to develop economically feasible, technically efficient and environmentally friendly method.

6. New Pretreatment Methods
Unique solvent properties
Ionic Liquid
Fluidity
Minimum volatility
Recyclable
Inability to use as fuel
Byproduct of biodiesel
Crude Glycerol
Purification not feasible
High B.P.
Possibility for process modification
No of unit operations at the same time
Extrusion Cooking
No fermentation inhibitors
High shearing force

7. FeedStocks
Cost and availability of feedstock is also equally important
Potential Feedstocks:
Water Hyacinth Lignocellulosic Waste
Rapid growth rate
73% wt gain/wk and yield up to 154 tons/ha-year
Invasive aquatic pest
Major Problem of waterways
No requirement of land
Additional benefit: - Water Purification.
Wheat Straw Agricultural residue
Agricultural residue of one of the major World’s crops

8. Agenda Background of Study Objectives Materials & Methods
Results & Discussion
Conclusion & Recommendations

9. Objective
To evaluate and compare the effectiveness of Crude Glycerol, Ionic Liquid and Extrusion Cooking for the pretreatment of wheat straw and water hyacinth.
Reference Methods: - Dilute acid pretreatment for all methods
Pure glycerol pretreatment for crude glycerol
Measure of effectiveness:
Enzymatic hydrolysis yield of glucose and total reducing sugar using cellulase from Trichoderma reesei.
Fermentation yield of ethanol using Saccharomyces cerevisiae.

10. Agenda Background of Study Objectives Materials & Methods
Results & Discussion
Objectives
Conclusion & Recommendations

11. Materials 1. Feedstocks Water Hyacinth Wheat Straw 2. Crude Glycerol
Collected from canal of AIT
Washing , chopping, drying
Grinding (sieve size: µm)
Wheat Straw
Collected from farm in Dijon (France)
Grinding (sieve size: - 1 cm, followed by 750 µm)
2. Crude Glycerol
From a biodiesel industry in France

12. 3. Ionic Liquids 4. Extruder 5. Enzyme 6. Yeast
1-butyl-3-methylimidazolium acetate, (BMIMA)
1-ethyl-3-methylimidazolium diethyl phosphate (EMIMDP)
4. Extruder
AgroSup (also called ENSBABA) laboratory, Dijon, France
5. Enzyme
Cellulase from Trichoderma reesei
6. Yeast
Saccharomyces cerevisiae

13. Methodology Overall work in 6 sequential steps
1. Optimization of enzyme concentration and hydrolysis time
2. Selection of the best condition for Crude Glycerol and Ionic Liquid Pretreatment
3. Selection of the best condition for Extrusion Cooking Pretreatment
4. Comparison of different pretreatment methods, at the selected condition, for hydrolysis yield of glucose and TRS
5. Optimization of fermentation time and initial glucose conc. for fermentation medium
6. Comparison of different pretreatment methods for fermentation yield of ethanol
INRA, France
AIT

14. Agenda Background of Study Objectives Materials & Methods
Results & Discussion
Objectives
Conclusion & Recommendations

15. 4. Results and Discussion
1 Optimization of enzyme concentration and hydrolysis time
(1µL = 0.84 units cellulase)
Hydrolysis yield of glucose at different enzyme concentration from wheat straw.
Results
Optimum Incubation time: less than 3 h (2.5 h)
Optimum enzyme concentration: 250 µl/100 mg sample

16. Method of Pretreatment
2 Selection of the best pretreatment condition
2.1 Crud Glycerol (C.G.) and Ionic Liquid (I.L.) Pretreatments
22 Factorial Design was used (Minitab for data analysis).
Maximum temp and time were fixed
C.G ºC for 4 h
Difficult to control beyond this due to excessive foam formation
I.L ºC for 1 h
Difficult to stir during heating beyond this due to thickening of solution
Method of Pretreatment
Factor
Level
Minimum
Maximum
Crude Glycerol
Time (h) 1 4
Temperature (oC)
180
230
Ionic Liquids
Time (min) 10 60
100
150

17. Table Hydrolysis Yield after Crude Glycerol Pretreatment at Different Conditions
Pretreatment Condition
(Time and Temp)
Wheat Straw Sample
Water Hyacinth Sample
Glucose (mg/g pretreated sample)
Reducing Sugar (mg/g pretreated sample)
1 (at 205oC for 2.5 h)
172
329
450
710
2 (at 205oC for 2.5 h)
164
307
462
742
3 (at 205oC for 2.5 h)
185
338
461
706
4 (at 230oC for 1 h)
186
332
451
705
5 (at 180oC for 1 h) 79
130
167
227
6 (at 180oC for 4 h)
132
261
388
592
7 (at 230oC for 4 h)
238
423
436
696

18. Table For BMIMA (an ionic liquid) Pretreatment
Pretreatment Condition
(Time and Temp)
Wheat Straw Sample
Water Hyacinth Sample
Glucose (mg/mg pretreated sample)
Reducing Sugar (mg/mg pretreated sample)
1 (at 125oC for 35 min)
352
664
274
361
2 (at 125oC for 35 min)
332
642
234
339
3 (at 125oC for 35 min)
320
568
247
353
4 (at 150oC for 10 min)
359
677
292
460
5 (at 100oC for 10 min)
120
214
198
261
6 (at 100oC for 60 min)
201
341
240
314
7 (at 150oC for 60 min)
368
744
584
Table For EMIMDP (an ionic liquid) Pretreatment
Pretreatment Condition
(Time and Temp)
Wheat Straw Sample
Glucose (mg/mg pretreated sample)
Reducing Sugar (mg/mg pretreated sample)
1 (at 125oC for 35 min)
103
168
2 (at 125oC for 35 min)
158
3 (at 125oC for 35 min) 98
170
4 (at 150oC for 10 min)
120
208
5 (at 100oC for 10 min) 78
119
6 (at 100oC for 60 min) 76
126
7 (at 150oC for 60 min)
148
283

19. 2.2 Extrusion Cooking Pretreatments: for wheat straw
Crude glycerol and pure glycerol: -additives
100, 150, 200 and 220 % of sample.
Screw speed: 150 and 300 rpm
Barrel temperature:
at 40ºC - material blocked in extruder
at 80ºC –blocked at 150 rpm.
Other temp- 120 and140ºC.
Carried out 13 different experiments.
Results
No significant difference in hydrolysis yield among tested conditions
Very low yield compared to other methods – less than 100 mg/g sample.

20. 3 Comparison of Enzymatic Hydrolysis Yield
3.1 Wheat Straw
744
487
423
Reference Method
283
223 99
Ionic Liquids
Figure 7 Hydrolysis yield of glucose and total reducing sugar of wheat straw samples pretreated by different pretreatment methods.

21. 3.2 Water Hyacinth
Reference method
Figure 8Hydrolysis yield of glucose and total reducing sugar of water hyacinth samples pretreated by different pretreatment methods.

22. 4 Optimization of Fermentation Time and Initial Glucose Conc
4 Optimization of Fermentation Time and Initial Glucose Conc. in Fermentation Media
WS: - hydrolysate of wheat straw sample; WH: - hydrolysate of water hyacinth sample;
Pure Glucose: - glucose solution in acetate buffer; mg/ml: - concentration of glucose
Results
6 h fermentation time with 12 to 18 mg/ml initial glucose conc. is optimum.

23. 5 Comparison of Fermentation Yield
4.5.1 Wheat Straw
Reference method
control
EMIMDP
Figure 12 Yield of ethanol (mg/mg glucose).
Figure 13 Glucose Conc. (mg/ml) in media

24. 4.5.1 Water Hyacinth Figure 14 Yield of ethanol (mg/mg glucose).
Ref Method
Control
Figure 14 Yield of ethanol (mg/mg glucose).
Figure 15 Glucose Conc. (mg/ml) in media

25. Agenda Background of Study Objectives Materials & Methods
Results & Discussion
Objectives
Conclusion & Recommendations

26. Overall Conclusion Wheat Straw Water Hyacinth
BMIMA ( a ionic liquid) is the best method: - 3 times more hydrolysis yield than dil. acid
Crude glycerol also better than dil. Acid: – double yield than dil. acid
Water Hyacinth
Crude glycerol is the best method of pretreatment
Crude glycerol is as effective as pure glycerol for both feedstocks.
Crude glycerol could be superior to ionic liquids even for wheat straw if current high cost of ionic liquids and byproduct utilization of biodiesel are taken into consideration.

27. Recommendations Further research work is needed to:
To study the recovery of ionic liquid and the effectiveness of the recycled ionic liquids.
To explore the possibility of enzymatic hydrolysis of crude glycerol pretreated sample without washing, followed by co-fermentation of the hydrolysates using glycerol as well as pentose and hexose fermenting microorganisms.
To evaluate cost of bioethanol when the water hyacinth plant is utilized simultaneously for bioethanol production as well as animal feed supplement production.

28. Thank you
for
Your kind Attention

29. Specific Objectives Objective 1 Objective 2 Objective 3
Crude Glycerol
Selection of the best condition of pretreatment
Ionic Liquids
Extrusion Cooking
Objective 2
Comparison of Pretreatment methods on the basis of hydrolysis yield
Crude Glycerol
Dilute Acid
Ionic Liquids
Extrusion Cooking
Pure Glycerol
Objective 3
Comparison of these Pretreatment methods on the basis of fermentation yield

30. Scope and Limitation Assumption
Focused on cellulose hydrolysis and glucose fermentation only
Hydrolysis yield on the basis of pretreated sample.
Assumption
Fig. Simplified structure of cellulose-hemicelluloses-lignin Complex
A pretreatment method which leads better improvement of enzymatic hydrolysis of cellulose would obviously improve hydrolysis of hemicelluloses.
Cellulose
Hemicelluloses
Lignin

31. Similar result for liberation of TRS from wheat straw sample as well as glucose and TRS from Water hyacinth sample.
Fig. 4.2 TRS from wheat straw
Fig. 4.3 Glucose from water hyacinth
Fig. 4.4 TRS from water hyaicnth
Results
For comparative study,
Optimum incubation time h
→ reaction rate is linear till 3 h
optimum enzyme conc µl/100 mg sample
→ Sugar conc. increase from 50 to 250, but not 250 to 500

32. 1. Optimization of enzyme concentration and hydrolysis time
Pretreatment of both biomass samples using crude glycerol at 230ºC for 4 h (10g:200ml)
Washed with water and dried
Hydrolysis of pretreated sample with various enzyme loading and incubation time, at 50ºC
Determination of Glucose and TRS
Graphs: - Sugar conc. Vs Incubation time, at various enzyme conc.
Crude Glycerol Pretreatment
Determination of optimum enzyme conc. and hydrolysis time

33. 2. Selection of the best condition for Crude Glycerol Pretreatment (Objective 1)
Mixed 10 mg sample in 200 ml crude glycerol
Heated at various temp and time
(180 to 230ºC for 1 to 4 h)
Washed with water and dried
Hydrolysis (250µl enz/100 mg sample at 50ºC for 2.5 h)
Determination of Glucose and TRS
Analysis of data
Ionic Liquid Pretreatment
Determination of optimum pretreatment condition using Crude Glycerol for each biomass sample
Crude Glycerol Pretreatment

34. 3. Selection of the best condition for Ionic Liquid Pretreatment (Objective 1)
Dissolved 240 mg sample in 6 g ionic liquid
Heated at various temp and time
(100 to 150ºC for 10 to 60 min)
Washed with water and dried
Hydrolysis (250µl enz/100 mg sample at 50ºC for 2.5 h)
Determination of Glucose and TRS
Analysis of data
Ionic Liquid Pretreatment
Determination of optimum pretreatment condition using Ionic liquid for each biomass sample

35. 4. Selection of the best condition for Extrusion Cooking Pretreatment (Objective 1)
Mixed sample with additives (100 to 220% of sample)
Extruded at various screw speed and barrel temperature
Washed with water and dried
Hydrolysis (250µl enz/100 mg sample at 50ºC for 2.5 h)
Determination of Glucose and TRS
Analysis of data
Extrusion Cooking Pretreatment
Determination of optimum pretreatment condition using Extrusion Cooking for wheat straw sample

36. 5. Comparison of different pretreatment methods, at the selected condition, for hydrolysis yield of glucose and TRS (Objective 2)
Pretreatment of sample using all methods at the selected condition (including dil. acid and pure glycerol pretreatments)
Washed with water and dried
Hydrolysis (250µl enz/100 mg sample at 50ºC for 2.5 h)
Determination of Glucose and TRS
Analysis of data
Determination of the best method of pretreatment for each biomass sample

37. 6. Optimization of fermentation time and initial glucose conc
6. Optimization of fermentation time and initial glucose conc. for fermentation medium
Hydrolysates of dil. acid pretreated sample with different glucose concentration
Preparation of fermentation media, adding nutrient supplements
Inoculation of inoculums of Saccharomyces cerevisiae
Fermentation at 30ºC
Determination of ethanol, glucose and TRS at various time intervals
Plotted ethanol conc. Vs fermentation time for various initial glucose conc
Determination of optimum fermentation time and initial glucose conc. for fermentation medium

38. 7. Comparison of different pretreatment methods for fermentation yield of ethanol (Objective 3)
Hydrolysates of samples, pretreated by different methods, with 12 to 15 mg/ml glucose concentration (including control)
Preparation of fermentation media, adding nutrient supplements
Inoculation of inoculum of Saccharomyces cerevisiae
Fermentation at 30ºC for 6 h or more
Determination of ethanol, glucose and TRS at various time intervals
Analysis of data, statistically as well as graphically
Determination of best method of pretreatment for each biomass sample

39. 2.2 Extrusion Cooking Pretreatments
Table Extrusion Parameters used and Hydrolysis Yield Wheat Straw Samples
Additive used
Amount of additive
(% of Sample)
Barrel Temp (oC)
Screw Speed (rpm)
Observation
Glucose Yield
(mg/mg sample)
TRS Yield
*C. G.
200
120
300
Compact, dark brown, slight expansion at the output 57 87
C. G.
150
Higher expansion at the output than Outside brown, but not inside 63 98
140
Same as 1001 59 84
More compact, lower expansion. 62
Expansion at the output same as 1000 60 95
100
More viscous, no expansion 66 99
**P .G.
Slightly more expanded than 1400 56
101
P .G. 40
Blocked - 80
Dark brown, compact 54 83
220
Dark brown, very compact 46 74
Slight expansion at the output 81
Expansion at the output 90
*C.G. = Crude Glycerol, * * P.G. = Pure Glycerol (purity 99%)

40. Wheat Straw
Method of Pretreatment
Glucose Yield
Reducing Sugar Yield
Yield (mg/g Sample)
Relative Yield
(%)
Yield (mg/mg Sample)
EMIMDP
0.15 ± 0.00 b 84
0.28 ± 0.00 c
127
BMIMA
0.37 ± 0.00 f
209
0.74 ± 0.01 f
334
Crude Glycerol
0.24 ± 0.03 d
135
0.42 ± 0.02 d
190
Pure Glycerol
0.26 ± 0.01 e
148
0.49 ± 0.01 e
219
Extrusion
0.07 ± 0.01a 38
0.10 ± 0.02 a 45
Dilute Acid
0.18 ± 0.01 c
100
0.22 ± 0.01 b
Water Hyacinth
Method of Pretreatment
Glucose Yield
Reducing Sugar Yield
Yield (mg/g Sample)
Relative Yield
(%)
Yield (mg/mg Sample)
EMIMDP
0.26 ± 0.03 a 57
0.42 ± 0.01 a 59
BMIMA
0.33 ± 0.01 b 75
0.58 ± 0.02 b 82
Crude Glycerol
0.45 ± 0.03 c d
101
0.70 ± 0.02 c 99
Pure Glycerol
0.49 ± 0.01 d
110
0.72 ± 0.01 c
Extrusion

41. FigureYield of glucose and total reducing sugar of water hyacinth samples after two step acid hydrolysis.
Indication of almost absence of hemicelluloses in dil. acid, crude glycerol and pure glycerol pretreated samples.

42. Wheat Straw
Method of Pretreatment
Glucose in Hydrolysates (mg/ml)
Glucose Conc. in Ferm. Media (mg/ml)
Ethanol Yield
(mg/mg glucose)
Ethanol Yield (% of Theoretical Yield)
EMIMDP
7.33
15.57
0.38 ± 0.03a 75
BMIMA
10.93
15.44
0.38 ± 0.00a 74
Dilute Acid
13.60
16.48
0.39 ± 0.01a 76
Crude Glycerol
15.5
17.27
0.37 ± 0.01a 73
Pure Glycerol
13.22
15.8
0.37 ± 0.02a
Control -
13.95
0.37 ± 0.04a
Water Hyacinth
Method of Pretreatment
Glucose in Hydrolysates (mg/ml)
Glucose Conc. in Ferm. Media (mg/ml)
Ethanol Yield
(mg/mg glucose)
Ethanol Yield (% of Theoretical Yield)
EMIMDP
9.23
14.56
0.44 ± 0.02ab 86
BMIMA
10.25
14.94
0.42 ± 0.01a 82
Dilute Acid
28.14
12.27
0.47 ± 0.02b 92
Crude Glycerol
25.8
12.75
0.44 ± 0.01ab 87
Pure Glycerol
24.07
12.33
0.47 ± 0.03b
Control -
11.76
0.45 ± 0.01ab 88

43. BMIMA (an ionic liquid): - best method
Conclusion . 1. Comparison of pretreatment methods for Hydrolysis Yield
Wheat Straw
BMIMA (an ionic liquid): - best method
Glucose yield: times more that dil. acid (368 mg/g)
Total reducing sugar (TRS): times more than dil. acid (744 mg/g)
Crude Glycerol: - glucose: -1.4 and TRS: -1.9 times more than dil. acid
EMIMDP (an ionic liquid): - not as effective as BMIMA
- even less than crude glycerol
Extrusion Cooking: - ineffective (yield less than 0.1mg/mg)

44. Crude Glycerol: - best method
Water Hyacinth
Crude Glycerol: - best method
Glucose yield : mg/g and TRS yield: mg/g
As effective as pure glycerol and dil. acid
Ionic Liquid:
BMIMA: - Glucose -75% and TRS- 82% of dil. acid
EMIMDP: - Glucose-57% and TRS- 59% of dil. Acid

45. 2. Comparison of Pretreatment Methods for Fermentation Yield
Wheat Straw
No significant different among samples, except EMIMDP.
Highest: - Dilute acid →0.39 mg/mg glucose (76% of theoretical yield)
Lowest: - Pure glycerol and crude glycerol→ 0.37 mg/mg glucose (73% of theoretical)
Water hyacinth
No significant different among samples.
Highest: -Dilute acid and pure glycerol → mg/mg glucose (92% of theoretical yield)
Lowest: - BMIMA → 0.42 mg/mg glucose (82% of theoretical yield)