master thesis may 2010 new pretreatment methods for lignocellulosic residue for second generation...

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

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Hurdles1st generation (from sugar and starchy materials)

:Food security challenge:Limited feedstock2nd generation (from non-edible parts of

plants): Commercial production not economically

viable

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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

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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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