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 1 Background of Study 2 Objectives 3 Materials and Methods 4
Results and Discussion 5
Conclusion and 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
4. Distillation
A lot of challenges in each steps
Major bottleneck: - Pretreatment
Pure Ethanol

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. Objectives 2 1 Background of Study 3 Materials and Methods 4
Results and Discussion 5
Conclusion and 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. Materials and Methods 3 1 Background of Study 2 Objectives 4
Results and Discussion 5
Conclusion and 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. Results and Discussion1
Background of Study 2
Objectives 3
Materials and Methods 4
Results and Discussion 5
Conclusion and 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 of Glucose and Total Reducing Sugar 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
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 of Hydrolysates (Objective 3)
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 Control
Ref Method
Control
Figure 14 Yield of ethanol (mg/mg glucose).
Figure 15 Glucose Conc. (mg/ml) in media

25. 5 Conclusion and Recommendations 1 Background of Study 2 Objectives 3
Materials and Methods 4
Results and Discussion 5
Conclusion and Recommendations

26. Overall Conclusion
BMIMA ( a ionic liquid) is the best for wheat straw- 3 times more hydrolysis yield than dil. acid
Crude glycerol also better than dil. Acid – double yield
Crude glycerol is the best for water hyacinth
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