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AdviserProf. Sudip K. Rakshit
New Pretreatment Methods for Lignocellulosic Residue for Second Generation Bioethanol Production
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Master ThesisMay 2010
Student:Yadhu Nath Guragain
ID: -107587
Agenda
Background of Study
Materials & Methods
Results & Discussion
Objectives
Conclusion & Recommendations
Global Energy ScenarioDemand gradually Increasing
Major source, fossil fuel, DepletingClimate change (GHG emission)
Alarming
PossibilitySubstitution of gasoline by Bioethanol
Background of Study
3
Hurdles1st generation (from sugar and starchy materials)
:Food security challenge:Limited feedstock2nd generation (from non-edible parts of
plants): Commercial production not economically
viable
4
1. Pretreatment
Cellulose
Lignin
Hemicelluloses
Lignocellulose
Monomer Sugars
2. Hydrolysis
3. Fermentation
Ethanol (ca. 10%)
Pure Ethanol (ca. 99.5%)
4. Distillation
Production Process of 2nd Generation Bioethanol
A lot of challenges in each stepsMajor bottleneck: - Pretreatment
Pretreatment
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.
Objective of pretreatmentTo break down lignin-hemicelluloses-cellulose complex, making carbohydrate
polymers susceptible for enzymatic hydrolysis
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New Pretreatment Methods
Ionic Liquid
Unique solvent properties
RecyclableMinimum volatility
Fluidity
Crude Glycerol
Byproduct of biodiesel
Inability to use as fuel
Purification not feasibleHigh B.P.
Extrusion Cooking
Possibility for process modification
High shearing force
No of unit operations at the same time
No fermentation inhibitors
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FeedStocksCost and availability of feedstock is also equally important
Potential Feedstocks:
1. 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.
2. Wheat Straw Agricultural residue Agricultural residue of one of the major World’s crops
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Agenda
Background of Study
Materials & Methods
Results & Discussion
Objectives
Conclusion & Recommendations
ObjectiveTo 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.
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Agenda
Background of Study
Materials & Methods
Results & Discussion
Objectives
Conclusion & Recommendations
Materials1. Feedstocks
I. Water Hyacinth
Collected from canal of AIT
Washing , chopping, drying
Grinding (sieve size: - 750 µm)
II. 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
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3. Ionic Liquids 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
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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
13
INR
A, F
ranc
eA
IT
Agenda
Background of Study
Materials & Methods
Results & Discussion
Objectives
Conclusion & Recommendations
4. Results and Discussion1 Optimization of enzyme concentration and hydrolysis time
0 5 10 15 20 250.000.050.100.150.200.250.300.350.400.45
50 uL
100 uL
250 uL
500 uL
Incubation Time (h)
Glu
cose
Yie
ld (m
g/m
g sa
mpl
e)
Hydrolysis yield of glucose at different enzyme concentration from wheat straw.
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(1µL = 0.84 units cellulase)
ResultsOptimum Incubation time: less than 3 h (2.5 h)Optimum enzyme concentration: 250 µl/100 mg sample
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. 230ºC for 4 hDifficult to control beyond this due to excessive foam formation
I.L. 150ºC for 1 h Difficult to stir during heating beyond this due to thickening of
solution
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Method of Pretreatment Factor
Level
Minimum Maximum
Crude Glycerol Time (h) 1 4
Temperature (oC) 180 230
Ionic LiquidsTime (min) 10 60
Temperature (oC) 100 150
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)
Glucose (mg/g pretreated sample)
Reducing Sugar (mg/g pretreated
sample)1 (at 205oC for 2.5 h) 172 329 450 7102 (at 205oC for 2.5 h) 164 307 462 7423 (at 205oC for 2.5 h) 185 338 461 7064 (at 230oC for 1 h) 186 332 451 7055 (at 180oC for 1 h) 79 130 167 2276 (at 180oC for 4 h) 132 261 388 5927 (at 230oC for 4 h) 238 423 436 696
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Table For BMIMA (an ionic liquid) Pretreatment
Pretreatment Condition (Time and Temp)
Wheat Straw Sample Water Hyacinth SampleGlucose (mg/mg
pretreated sample)Reducing Sugar (mg/mg
pretreated sample)Glucose (mg/mg
pretreated sample)Reducing Sugar (mg/mg
pretreated sample)
1 (at 125oC for 35 min) 352 664 274 3612 (at 125oC for 35 min) 332 642 234 3393 (at 125oC for 35 min) 320 568 247 3534 (at 150oC for 10 min) 359 677 292 4605 (at 100oC for 10 min) 120 214 198 2616 (at 100oC for 60 min) 201 341 240 3147 (at 150oC for 60 min) 368 744 332 584
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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 1682 (at 125oC for 35 min) 103 1583 (at 125oC for 35 min) 98 1704 (at 150oC for 10 min) 120 2085 (at 100oC for 10 min) 78 1196 (at 100oC for 60 min) 76 1267 (at 150oC for 60 min) 148 283
Table For EMIMDP (an ionic liquid) Pretreatment
Crude glycerol and pure glycerol: -additives100, 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.
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2.2 Extrusion Cooking Pretreatments: for wheat straw
3 Comparison of Enzymatic Hydrolysis Yield
3.1 Wheat Straw
Figure 7 Hydrolysis yield of glucose and total reducing sugar of wheat straw samples pretreated by different pretreatment methods. 20
EMIMDP BMIMA Crude Glycerol
Pure Glycerol
Extrusion Dil Acid0
100
200
300
400
500
600
700
800 Glucose YieldReducing Sugar Yield
Method of Pretreatment
Suga
r Yie
ld (m
g/g
sam
ple)
Reference Method
744
223
487
99
423
283
Ionic Liquids
3.2 Water Hyacinth
Figure 8Hydrolysis yield of glucose and total reducing sugar of water hyacinth samples pretreated by different pretreatment methods. 21
EMIMDP BMIMA Crude Glycerol
Pure Glycerol
Dil Acid0
100
200
300
400
500
600
700
800Glucose YieldReducing Sugar Yield
Method of Pretreatment
Suga
r Yie
ld (m
g/g
sam
ple)
422
705
584
719 714
Ionic Liquids
Reference method
Results6 h fermentation time with 12 to 18 mg/ml initial glucose conc. is optimum.
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WS: - hydrolysate of wheat straw sample; WH: - hydrolysate of water hyacinth sample; Pure Glucose: - glucose solution in acetate buffer; mg/ml: - concentration of glucose
4 Optimization of Fermentation Time and Initial Glucose Conc. in Fermentation Media
0 2 4 6 8 10 12 14 16 18 200.00
0.10
0.20
0.30
0.40
0.50WS-8 mg/ml
WH- 8 mg/ml
WH- 12 mg/ml
WH- 15 mg/ml
Pure Glucose- 18 mg/ml
Fermentation Time (h)
Etha
nol Y
ield
(mg/
mg
gluc
ose)
5 Comparison of Fermentation Yield
4.5.1 Wheat Straw
Figure 12 Yield of ethanol (mg/mg glucose).
0 5 10 15 20 25 300.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
Fermentation time (hr)
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0 2 4 6 8 10 12 1402468
101214161820
EMIMDPBMIMADilute AcidCrude GlycerolPure GlycerolControl
Fermentation Time (h)
Figure 13 Glucose Conc. (mg/ml) in media
control
Reference method
EMIMDP
4.5.1 Water Hyacinth
Figure 14 Yield of ethanol (mg/mg glucose).
0 2 4 6 8 10 12 140.000.050.100.150.200.250.300.350.400.450.50
Fermentation Time (h)
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0 1 2 3 4 5 6 70
2
4
6
8
10
12
14
16EMIMDP
BMIMA
Dilute Acid
Crude Glycerol
Pure Glycerol
Control
Fermentation Time (hr)
Figure 15 Glucose Conc. (mg/ml) in media
Control
Ref Method
Agenda
Background of Study
Materials & Methods
Results & Discussion
Objectives
Conclusion & Recommendations
Overall ConclusionWheat Straw
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.
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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.
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Thank you for
Your kind Attention
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