Master Thesis May 2010 New Pretreatment Methods for Lignocellulosic Residue for Second Generation Bioethanol Production Student: Yadhu Nath Guragain ID:

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Presentation transcript:

Master Thesis May 2010 New Pretreatment Methods for Lignocellulosic Residue for Second Generation Bioethanol Production Student: Yadhu Nath Guragain ID: -107587 Adviser Prof. Sudip K. Rakshit

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

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

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%)

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.

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

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

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

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.

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

Materials 1. Feedstocks Water Hyacinth Wheat Straw 2. Crude Glycerol Collected from canal of AIT Washing , chopping, drying Grinding (sieve size: - 750 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

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

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

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

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

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. 230ºC for 4 h Difficult 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 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

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

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

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.

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.

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

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.

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

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

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

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.

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.

Thank you for Your kind Attention

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

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

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 2.5 h → reaction rate is linear till 3 h optimum enzyme conc. 250 µl/100 mg sample → Sugar conc. increase from 50 to 250, but not 250 to 500

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

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

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

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

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

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

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

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 1000. 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%)

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

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.

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

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: - 2.1 times more that dil. acid (368 mg/g) Total reducing sugar (TRS): - 3.3 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)

Crude Glycerol: - best method Water Hyacinth Crude Glycerol: - best method Glucose yield : - 451 mg/g and TRS yield: - 705 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

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 → 0.47 mg/mg glucose (92% of theoretical yield) Lowest: - BMIMA → 0.42 mg/mg glucose (82% of theoretical yield)