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Biodiesel Technology
and Applications
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Biodiesel Technology
and Applications
Edited by
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Contents
Preface xvii
1 Biocatalytic Processes for Biodiesel Production 1
Ubaid Mehmood, Faizan Muneer, Muhammad Riaz,
Saba Sarfraz and Habibullah Nadeem
1.1 Introduction and Background 2
1.2 Importance of Biodiesel Over Conventional Diesel Fuel 3
1.3 Substrates for Biodiesel Production 4
1.4 Methods in Biodiesel Production 6
1.5 Types of Catalysts Involved in Biodiesel Production 7
1.5.1 Chemical Homogenous Catalysts 7
1.5.2 Solid Heterogeneous Catalysts 8
1.5.3 Biocatalysts 8
1.6 Factors Affecting Enzymatic Transesterification Reaction 8
1.6.1 Effect of Water in Enzyme Catalyzed
Transesterification 9
1.6.2 Effect of Bioreactor 10
1.6.3 Effect of Acyl Acceptor on Enzymatic Production
of Biodiesel 10
1.6.4 Effect of Temperature on Enzymatic Biodiesel
Production 14
1.6.5 Effect of Glycerol on Enzymatic Biodiesel Production 14
1.6.6 Effect of Solvent on Biodiesel Production 16
1.7 Lipases as Biocatalysts for Biodiesel Production 17
1.7.1 Mechanisms of Lipase Action 19
1.7.2 Efficient Lipase Sources for Biodiesel Producing
Biocatalyst 19
1.8 Comparative Analysis of Intracellular and Extracellular
Lipases for Biodiesel Production 21
1.9 Recombinant Lipases for Cost-Effective Biodiesel Production 26
1.10 Immobilization of Lipases for Better Biodiesel Production 28
v
vi Contents
1.11 Recent Strategies to Improve Biodiesel Production 31

1.11.1 Combination of Lipases 31
1.11.2 Microwave and Ultrasonic-Assisted Reaction 33
1.12 Lipase Catalyzed Reaction Modeling and Statistical
Approaches for Reaction Optimization 35
1.13 Conclusion and Summary 38
References 38
2 Application of Low-Frequency Ultrasound for Intensified
Biodiesel Production Process 59
Mohd Razealy Anuar, Mohamed Hussein Abdurahman,
Nor Irwin Basir and Ahmad Zuhairi Abdullah
2.1 Current Fossil Fuel Scenario 60
2.2 Biodiesel 60
2.3 Transesterification 61
2.4 Challenges for Improved Biodiesel Production 62
2.5 Homogeneous Catalyst for Biodiesel Production 63
2.6 Heterogeneous Catalyst for Biodiesel Production 64
2.7 Immiscibility of the Reactants 65
2.8 Ultrasound-Assisted Biodiesel Production Process 66
2.8.1 Fundamental Aspects of the Process 66
2.8.2 Homogeneously Catalyzed Ultrasound-Assisted
System 69
2.8.3 Heterogeneously Catalyzed Ultrasound-Assisted
System 72
2.8.3.1 Heterogeneously Acid Catalyzed System 72
2.8.3.2 Heterogeneous Based Catalyzed
Ultrasound-Assisted System 74
2.8.3.3 Influence of Reaction Parameters 78
2.9 Conclusions 79
Acknowledgement 80
References 80
3 Application of Catalysts in Biodiesel Production 85
Anilkumar R. Gupta and Virendra K. Rathod
3.1 Introduction 85
3.2 Homogeneous Catalysis for the Biodiesel Production 89
3.2.1 Homogeneous Acid Catalyst 89
3.2.2 Homogeneous-Base Catalyst 93
3.3 Heterogeneous Catalyst 96
3.3.1 Heterogeneous Acid Catalyst 97
3.3.2 Heterogeneous-Base Catalyst 106
Contents vii
3.4 Biocatalysts 115

3.5 Conclusion 119
References 124
4 Hydrogenolysis as a Means of Valorization
of Biodiesel-Derived Glycerol: A Review 137
Manjoro T.T., Adeniyi A. and Mbaya R.K.K.
4.1 Introduction 138
4.2 Ways of Valorization of Biodiesel-Derived Glycerol 139
4.2.1 Catalytic Conversion of Glycerol Into Value-Added
Commodities 140
4.2.1.1 Catalytic Oxidation of Glycerol 140
4.2.1.2 Catalytic Dehydration of Glycerol 143
4.2.1.3 Pyrolysis of Bioglycerol 144
4.2.1.4 Glycerol Transesterification 145
4.2.1.5 Glycerol Direct Carboxylation 146
4.3 Hydrogenolysis of Glycerol 147
4.3.1 Definition of Hydrogenolysis 147
4.3.2 Catalytic Hydrogenolysis of Glycerol 148
4.3.3 Product Spectrum from Hydrogenolysis of Glycerol 148
4.3.4 Hydrogenolysis of Glycerol to 1,2-PDO (Propylene
Glycol): Reaction Systems Overview 149
4.3.5 Catalyst Selection 151
4.3.6 Reaction Conditions That Influence the
Hydrogenolysis of Glycerol to 1,2-PDO 153
4.3.6.1 Effect of Reaction Temperature 153
4.3.6.2 Effect of H2 Pressure 154
4.3.6.3 Effect of Initial Water Concentration 155
4.3.6.4 Effect of Reaction Time 156
4.3.6.5 Effect of Catalyst Weight 156
4.3.6.6 Proposed Reaction Mechanisms for Glycerol
Hydrogenolysis to Produce 1,2-PDO 157
4.4 Conclusion 159
References 159
5 Current Status, Synthesis, and Characterization of Biodiesel 167
Akshay Garg, Gaurav Dwivedi, Prashant Baredar
and Siddharth Jain
5.2 Status of Biodiesel in India 169
5.3 Biodiesel Production in India 169
viii Contents
5.3.1 Feedstocks Popular in India 169

5.3.1.1 Jatropha (Jatropha curcas) Oil 171
5.3.1.2 Pongamia Oil 171
5.3.1.3 Mahua Oil 171
5.3.1.4 Neem Oil 171
5.3.1.5 Linseed Oil 171
5.3.1.6 Rubber Seed Oil 172
5.3.1.7 Tobacco Oil 172
5.3.1.8 Castor 172
5.3.1.9 Waste Cooking Oil 172
5.3.1.10 Algae Oil 172
5.3.2 Advantages of Non-Edible Oils 173
5.3.3 Modification Techniques 173
5.3.3.1 Blending 173
5.3.3.2 Micro-Emulsification 173
5.3.3.3 Cracking 174
5.3.3.4 Transesterification 174
5.3.4 Biodiesel Production Methodology 174
5.3.4.1 Catalytic Transesterification 174
5.3.4.2 Non-Catalytic Transesterification 178
5.3.5 Optimization Methodology for Biodiesel 179
5.3.5.1 Central Composite Design Technique 179
5.3.5.2 Box Behnken Technique 179
5.4 Properties of Biodiesel 180
5.5 Analytical Methods 181
5.5.1 Titration 181
5.5.2 Chromatic Methods 181
5.5.2.1 Gas Chromatography 183
5.5.2.2 High-Performance Liquid Chromatography 184
5.5.3 Spectroscopic Methods 184
5.5.3.1 Nuclear Magnetic Resonance Spectroscopy 184
5.5.3.2 Infrared Spectroscopy 185
5.5.4 Rancimat Method 185
5.5.5 Viscometry 186
5.6 Conclusion 186
References 187
6 Commercial Technologies for Biodiesel Production 195
Chikati Roick, Leonard Okonye, Nkazi Diankanua
and Gorimbo Joshua
Abbreviation 196
Contents ix

6.2 Biodiesel Production 197
6.3 Technologies Used for Biodiesel Production 198
6.3.1 Chemical Reaction (Transesterification) 199
6.3.2 Thermochemical Conversion 199
6.3.3 Biomechanical Conversion 201
6.3.4 Direct Combustion 201
6.4 Other Technologies in Use for Biodiesel Production 201
6.5 Feedstock Requirement 203
6.6 Some Problems Facing Commercialization of Biodiesel
in Africa 203
6.7 Case Studies/Current Status and Future Potential 204
6.8 Conclusions 207
Acknowledgments 208
References 208
7 A Global Scenario of Sustainable Technologies and Progress
in a Biodiesel Production 215
M. B. Kumbhar, P. E. Lokhande,, U. S. Chavan
and V.G. Salunkhe
7.2 Current Status of Feedstock for Biodiesel Production
Technology 218
7.3 Scenario of Biodiesel in Combustion Engine 222
7.4 Biodiesel Production Technologies 223
7.4.1 Direct Blending 223
7.4.2 Pyrolysis 224
7.4.3 Microemulsification 225
7.4.4 Transesterification 226
7.5 Microwave-Mediated Transesterification 227
7.6 Ultrasound-Mediated Transesterification 229
7.7 Catalysis in Biodiesel Production 230
7.7.1 Homogeneous Catalysts 230
7.7.2 Heterogeneous Catalysts 231
7.7.3 Heterogeneous Nanocatalysts 232
7.7.4 Supercritical Fluids 232
7.7.5 Biocatalysts 232
7.8 The Concept of Biorefinery 234
7.9 Summary and Outlook 236
7.10 Conclusion 237
References 237
x Contents
8 Biodiesel Production Technologies 241

Moina Athar and Sadaf Zaidi
8.2 Biodiesel Feedstocks 242
8.2.1 Selection of Feedstocks 243
8.3 Biodiesel Production Technologies 248
8.3.1 Pyrolysis 248
8.3.2 Dilution 249
8.3.3 Micro-Emulsion 249
8.3.4 Transesterification 249
8.3.4.1 Homogeneously Catalyzed
Transesterification Processes 250
8.3.4.2 Heterogeneously Catalyzed
Transesterification Processes 252
8.3.4.3 Enzymatic Catalyzed Transesterification
Processes 252
8.4 Intensification Techniques for Biodiesel Production 253
8.4.1 Supercritical Alcohol Method 253
8.4.2 Microwave Heating 253
8.4.3 Ultrasonic Irradiation 255
8.4.4 Co-Solvent Method 256
8.5 Other Techniques of Biodiesel Production 256
References 257
9 Methods for Biodiesel Production 267
M.Gul, M.A. Mujtaba, H.H. Masjuki, M.A. Kalam
and N.W.M. Zulkifli
9.1 Selection of Feedstock for Biodiesel 267
9.1.1 First-Generation Feedstock 268
9.1.2 Second-Generation Feedstock 268
9.1.3 Third-Generation Feedstock 269
9.2 Methods for Biodiesel Production 269
9.2.1 Dilution With Hydrocarbons Blending 269
9.2.2 Micro-Emulsion 269
9.2.3 Pyrolysis (Thermal Cracking) 270
9.2.4 Transesterification (Alcoholysis) 271
9.2.4.1 In Situ Transesterification
(Reactive Extraction) 271
9.2.4.2 Conventional Transesterification 272
9.2.4.3 Microwave/Ultrasound-Assisted
Transesterification 278
Contents xi
9.2.4.4 Variables Affecting Transesterification

Reaction 278
References 282
10 Non-Edible Feedstock for Biodiesel Production 285
Chikati Roick, Kabir Opeyemi Otun, Nkazi Diankanua
and Gorimbo Joshua
List of Abbreviations 286
10.2 Reports Relevant to Global Warming
and Renewable Energy 287
10.3 Biofuels as an Alternative Energy Source 288
10.3.1 First-Generation Biofuels 288
10.3.2 Second-Generation Biofuels 289
10.3.3 Third-Generation Biofuels 290
10.4 Benefits of Using Biodiesel 290
10.5 Technologies of Biodiesel Production From Non-Edible
Feedstock 291
10.6 Biodiesel Production by Transesterification 292
10.7 Non-Edible Feedstocks for Biodiesel Production 295
10.7.1 Non-Edible Vegetable Oils 296
10.7.2 Waste Cooking Oil 297
10.7.3 Algal Oil 298
10.7.4 Waste Animal Fat/Oil 299
10.8 Fuel Properties of Biodiesel Obtained From Non-Edible
Feedstock 299
10.9 Advantages of Non-Edible Feedstocks 302
10.10 Economic Importance of Biodiesel Production 302
10.11 Conclusions 303
Acknowledgments 303
References 304
11 Oleochemical Resources for Biodiesel Production 311
Gayathri R., Ranjitha J. and Vijayalakshmi Shankar
11.2 Definition of Oleochemicals 312
11.3 Oleochemical Types 313
11.4 Production of Biodiesel 315
11.5 Types of Feedstocks 317
11.5.1 Non-Edible Feedstocks 317
11.5.2 Non-Edible Vegetable Oil 317
xii Contents
11.5.3 Tall Oil 318

11.5.4 Waste Cooking Oils 318
11.5.5 Animal Fats 318
11.5.6 Chicken Fat 319
11.5.7 Lard 319
11.5.8 Tallow 320
11.5.9 Leather Industry Solid Waste Fat 321
11.5.10 Fish Oil 322
11.6 Uses of Oleochemicals 322
11.6.1 Polymer Applications 322
11.6.2 Application of Plant Oil as a Substitute
for Petro-Diesel 323
11.6.3 Used as Surfactants 323
11.6.4 Oleochemicals Used in Pesticide 324
11.6.5 Oleochemicals Used in Spray Adjuvants
and Solvents 324
11.7 Methyl Ester or Biodiesel Production 324
11.7.1 Palm Oil 326
11.7.2 Sunflower Oil 326
11.7.3 ME From AFW 327
11.8 Parameters Affecting the Yield of Biodiesel 327
11.8.1 Reaction Conditions 327
11.8.2 Catalyst 327
11.8.2.1 Alkali Catalyst 327
11.8.2.2 Acid Catalyst 329
11.8.2.3 Biocatalyst 329
11.8.2.4 Heterogeneous Catalyst 329
11.8.2.5 ME Conversion by Supercritical
Method 329
11.8.3 Properties of Feedstock 330
11.8.3.1 Composition of FA 330
11.8.3.2 FFA 330
11.8.3.3 Heat 330
11.8.3.4 Presence of Unwanted Materials 330
11.8.3.5 Titer 332
11.8.4 Characteristic of Feedstock 332
11.9 Optimization of Reactions Conditions for High Yield
and Quality of Biodiesel 332
11.9.1 Pre-Treatment of Feedstock 332
11.9.1.1 Elimination of Water 332
11.9.1.2 Elimination of Insoluble Impurities 332
Contents xiii
11.9.1.3 Elimination of Unsaponiﬁables 333

11.9.2 Characterization and Selection of Feedstocks 333
11.9.3 Selection of Reaction Conditions 333
11.10 Oil Recovery 333
11.10.1 Alkaline Flooding Method 333
11.10.2 Additives 334
11.11 Quality Improvement of Biodiesel 334
11.11.1 Additives for Improving Combustion Ability 334
11.11.2 Additives for Enhancing the Octane Number 334
11.11.3 Additives for Improving the Stability 334
11.11.4 Additives to Enhance Cold Flow Property 334
11.11.5 Additives to Enhance Lubricity 335
11.11.6 Additives to Enhance Cetane Number 335
11.12 Conclusion 335
Abbreviations 335
References 336
12 Overview on Different Reactors for Biodiesel Production 341
V. C. Akubude, K.F. Jaiyeoba, T.F Oyewusi, E.C. Abbah,
J.A. Oyedokun and V.C. Okafor
12.2 Biodiesel Production Reactors 342
12.2.1 Batch Reactor 343
12.2.2 Continuous Stirred Tank Reactor 344
12.2.3 Fixed Bed Reactor 346
12.2.4 Bubble Column Reactor 347
12.2.5 Reactive Distillation Column 349
12.2.6 Hybrid Catalytic Plasma Reactor 350
12.2.7 Microreactors Technology 350
12.2.8 Oscillatory Flow Reactors 353
12.2.9 Other Novel Reactors 353
12.3 Future Prospects 354
12.4 Conclusion 354
References 354
13 Patents on Biodiesel 361
Azira Abdul Razak, Mohamad Azuwa Mohamed
and Darfizzi Derawi
13.2 Generation of Biodiesel 362
13.3 Development of Catalyst 363
xiv Contents
13.3.1 Homogeneous Catalyst 364

13.3.2 Heterogeneous Catalyst 364
13.4 Method Producing Biodiesel 365
13.4.1 Pre-Treatment Process 365
13.4.2 Direct Use and Blending of Oils 366
13.4.3 Esterification of FFA 366
13.4.4 Transesterification of TAG 367
13.4.5 Pyrolysis 368
13.5 Reactor’s Technology for Biodiesel Production 369
13.5.1 Continuous Stirred Tank Reactor 370
13.5.2 Fixed Bed Reactor 370
13.5.3 Micro-Mixer Reactor 371
13.6 Conclusion 372
References 372
14 Reactions of Carboxylic Acids With an Alcohol Over
Acid Materials 377
J.E. Castanheiro
14.2 Zeolites 378
14.3 SO3H as Catalyst 379
14.4 Metal Oxides 380
14.5 Heteropolyacids 382
14.6 Other Materials 384
References 385
15 Biodiesel Production From Non-Edible and Waste
Lipid Sources 389
Opeoluwa O. Fasanya, Aishat A. Osigbesan
and Onoriode P. Avbenake
15.2 Non-Edible Plant-Based Oils 394
15.2.1 Jatropha curcas 394
15.2.2 Calophyllum inophyllum 397
15.2.3 Mesua ferrea 397
15.2.4 Jojoba Oil 398
15.2.5 Azadirachta indica 398
15.2.6 Rubber Seed Oil 399
15.2.7 Ricinus communis as Feedstock (Castor Oil) 402
15.2.8 Other Non-Edible Oils 403
Contents xv
15.3 Waste Animal Fats 404

15.4 Expired and Waste Cooking Oils 405
15.5 Algae/Microalgae 406
15.6 Insects as Biodiesel Feedstock 411
15.7 Deacidification 414
15.8 Other Technologies 414
15.9 Conclusion 415
References 415
16 Microalgae for Biodiesel Production 429
Charles Oluwaseun Adetunji, Victoria Olaide Adenigba,
Devarajan Thangadura and Mohd Imran Ahamed
16.2 Physicochemical Properties of Biodiesel From Microalgae 431
16.3 Genetic Engineering/Techniques Enhancing Biodiesel
Production 432
16.4 Nanotechnology in Microalgae Biodiesel Production 434
16.5 Specific Examples of Biodiesel Production From
Microalgae 434
16.6 Methodology Involved in the Extraction of Algae 438
16.6.1 Chemical Solvents Extraction 439
16.6.2 Extraction by Supercritical Carbon Dioxide 439
16.6.3 Extraction Using Biochemical Techniques 439
16.6.4 Extraction Involving Direct Transesterification 440
16.6.5 Extraction Using Transesterification Techniques 440
16.7 Conclusion and Future Recommendation to Knowledge 440
References 441
17 Biodiesel Production Methods and Feedstocks 447
Setareh Heidari and David A. Wood
17.2 Biofuel Classification in Terms of Origin and
Technological Conversion of Raw Materials 449
17.3 Techniques Capable of Producing Biodiesel
on Commercial Scales 451
17.3.1 Direct and Blending Methods With the Aim
of Biodiesel Generation 452
17.3.2 Microemulsion Methods 452
17.3.3 Pyrolysis Methods 453
17.3.4 Transesterification Methods 453
17.4 Influential Parameters on Biodiesel Production 454
xvi Contents
17.4.1 The Choice of Transesterification Catalysts 454

17.4.2 Effects of Catalyst Characteristics on Biodiesel
Production Efficiency 454
17.5 Biodiesel Markets and Economic Considerations 455
17.6 Challenges Confronting Biodiesel Uptake 456
17.7 Corrosion and Quality Monitoring Issues for Biodiesel 457
References 458
18 Application of Nanoparticles for the Enhanced Production
of Biodiesel 465
Muhammad Hilman Mustapha, Akhsan Kamil Azizi,
Wan Nur Aini Wan Mokhtar and Mohamad Azuwa Mohamed
18.2 Solid Nanoparticles 466
18.3 Nanobioparticles/Nanobiocatalyst 471
18.4 Magnetic Nanoparticles 473
18.5 How Nanoparticles Enhanced Biodiesel Production? 475
18.6 Conclusion 477
References 477
Index 481
Preface
Energy technologies have attracted great attention due to the fast develop-
ment of sustainable energy. Biodiesel technologies have been identified as
the sustainable route through which overdependence on fossil fuels can be
reduced. Biodiesel has played a key role in handling the growing challenge
of a global climate change policy. Biodiesel is defined as the monoalkyl
esters of vegetable oils or animal fats. Biodiesel is a cost-effective, renew-
able, and sustainable fuel that can be made from vegetable oils and ani-
mal fats. Compared to petroleum-based diesel, biodiesel would offer a
non-toxicity, biodegradability, improved air quality and positive impact
on the environment, energy security, safe-to-handle, store and transport,
and so on. Biodiesels have been used as a replacement of petroleum diesel
in transport vehicles, heavy-duty trucks, locomotives, heat oils, hydrogen
production, electricity generators, agriculture, mining, construction, and
forestry equipment.
This book describes a comprehensive overview, covering a broad range
of topics on biodiesel technologies and allied applications. Chapters cover
history, properties, resources, fabrication methods, parameters, formula-
tions, reactors, catalysis, transformations, analysis, in situ spectroscopies,
key issues and applications of biodiesel technology. It also includes bio-
diesel methods, extraction strategies, biowaste utilization, oleochemical
resources, non-edible feedstocks, heterogeneous catalysts, patents, and
case-studies. Progress, challenges, future directions, and state-of-the-art
biodiesel commercial technologies are discussed in detail. This book is
an invaluable resource guide for professionals, faculty, students, chemical
engineers, biotechnologists, and environmentalists in these research and
development areas. This book includes the eighteen chapters and the sum-
maries are given as follows.
Chapter 1 details the biocatalytic production of biodiesel. Microbial
enzymes such as lipases act as biocatalysts in the transesterification pro-
cess of biodiesel production. Suitable and cost-effective feedstocks or
xvii
xviii Preface
substrates for biodiesel production including their percentage yields are

discussed. Factors that affect the enzymatic transesterification reaction are
also explained.
Chapter 2 addresses ultrasonic energy which can increase the interface
area while creating a thermal effect in heterogeneous biodiesel production
process to result in higher biodiesel yield. Fundamental understanding of
the improved reactant-catalyst interaction, the nature of the thermal effect,
favorable process behaviors, reaction kinetic, as well as the effect on bio-
diesel quality is particularly addressed.
Chapter 3 is about the study of different types of catalysts used for
biodiesel production. The classification of catalysts, advantages, and lim-
itations, along with their mechanism, is explained. The heterogeneous
catalysts’ synthetic methods and immobilization of biocatalyst are also dis-
cussed in detail.
Chapter 4 discusses various methods used to produce value-added
chemicals from biodiesel-derived glycerol. The main focus being is given
to hydrogenolysis as a transformative process to selectively produce 1,2-
propanediol and the advancements in biodiesel technologies. Furthermore,
knowledge gaps are highlighted based on extensive literature research on
the subject.
Chapter 5 discusses various techniques of synthesizing biodiesel and
review of various existing analytical technologies for characterization of
biodiesel. The chapter focuses on the current status of biodiesel in India,
i.e., using non-edible sources and future feasibility of developing new
methods of characterization to reduce the cost of biodiesel production.
Chapter 6 examines various established technologies available for the
production of biodiesel, viz., chemical reaction, direct combustion, ther-
mochemical conversion, and biomechanical conversion. Each technology
is apportioned to a certain type of feedstock. Case studies, current sta-
tus, and future potential of commercialization of biodiesel production in
Africa are also discussed.
There is a huge demand for sustainable biofuel production in coming
decades. The key challenges for biodiesel production are high FFA with the
desired level of yield, stability, optimized and flexible production, commer-
cialization of feedstock and environmentally friendly cycle. The collective
effort and commitment of research survey regard feedstocks and commer-
cialization of technology around the globe towards sustainable energy are
expressed in terms of accelerating the biofuel economy in Chapter 7.
Chapter 8 provides an overview of the available feedstocks, pro-
duction methods, and the benefits and constraints of using homoge-
neous, heterogeneous, and enzymatic catalysts for biodiesel. Some latest
Preface xix
intensification techniques to manage mass transfer restrictions of oil and

alcohol phases along with some production cost reduction measures are
also highlighted.
Chapter 9 discusses different types of feedstocks used for synthesizing
biodiesel and feedstock selection criteria. Moreover, all biodiesel produc-
tion methods (i.e., dilution with hydrocarbons blending, micro-emulsion,
pyrolysis, and transesterification) are also described in detail with their
advantages and disadvantages. The major focus is given to the various
transesterification methods. Production methods also include experimen-
tal setup layouts, all process parameters, reaction conditions, the latest
advancement in reaction processes, and their effects on biodiesel yield.
Chapter 10 reviews the potential use of non-edible feedstocks in the
production of biodiesel. Special attention is given to the types of feedstocks
available and their production pathways to biodiesel. The state-of-the-art
technology, the properties of the fuel produced, and the environmental
concerns of biofuels are also discussed.
Chapter 11 discusses the various types of oleochemicals and their usage.
Optimization and production of biodiesel derived from oleochemicals
and their properties are also discussed. The primary focus is given for the
advantage of oleochemicals to be used as a potential feedstock for biodiesel
production from the available literature.
Chapter 12 provides details about the different configurations of reac-
tors used in biodiesel production. There are two types, namely, batch and
continuous reactors. Recently, other improved configurations like micro-
reactors have emerged. This chapter also discusses the merits and demerits
of these reactors.
Chapter 13 highlights and discusses the international patents on bio-
diesel applications. This chapter reviews the recent patents on the generation
of biodiesel which depends on the feedstock used, catalysts development,
the latest method for biodiesel production, and reactor technology for the
biodiesel production.
Chapter 14 overviews different reactions between a carboxylic acid
(fatty acids) and alcohol (methanol and ethanol) over heterogeneous cata-
lysts, an important step in biodiesel production. The nature of solid materi-
als, like zeolites, heteropolyacids, materials with sulfonic groups, inorganic
mixed oxides, and clays towards biodiesel production is discussed.
Chapter 15 sheds light on inedible feedstock that could be utilized for
biodiesel production. Plant-based and non-plant feedstock are discussed.
The waste lipid sources which are unfit for consumption are also high-
lighted. The chemical composition, economic viability, and sustainability
of some of these feedstocks are equally explored.
xx Preface
Chapter 16 provides detailed information on the fabrication of biodiesel

from microalgae. Specific information on the physical properties, amount
of biodiesel production, and level of transesterification of biodiesel are dis-
cussed. The application of photobioreactors for the production of biodiesel
with the special consideration of several factors such as flow rate, tempera-
ture, light intensity, CO2 concentration, and time is highlighted. Several
techniques for the extraction of biodiesel such as supercritical CO2, physi-
cochemical, direct transesterification, chemical solvents, and biochemical
respectively are highlighted.
Chapter 17 discusses the biofuel classification in terms of origin and
technological conversion of raw materials. Techniques capable of produc-
ing biodiesel on commercial scales are also presented. Furthermore, influ-
ential parameters and their roles in biodiesel production are elaborately
covered. Finally, challenges and limitations confronting biodiesel uptake
are presented.
Chapter 18 mainly explicates the application of nanoparticle catalysis
for the high production of biodiesel. In particular, various types of catalyst
nanoparticles with different synthesis strategy and their roles in enhancing
the biodiesel production are discussed.
Inamuddin, Mohd Imran Ahamed, Rajender Boddula

and Mashallah Rezakazemi
1
Biocatalytic Processes for
Biodiesel Production
Ubaid Mehmood1, Faizan Muneer2, Muhammad Riaz3, Saba Sarfraz4
and Habibullah Nadeem2*
College of Chemistry, Chemical Engineering and Biotechnology,

1
Donghua University, China

2
Department of Bioinformatics and Biotechnology, Government College University
Faisalabad, Pakistan
3
Department of Food Sciences, University College of Agriculture, Bahauddin
Zakariya University, Multan, Pakistan
4
Department of Chemistry, Government College Women University Faisalabad,
Faisalabad, Pakistan
Abstract
Enzymes such as microbial lipases can be effectively used as biocatalysts for bio-
diesel production in a sustainable manner. Biocatalytic processes to produce bio-
diesel or biofuel is the need of time to reduce the emission of greenhouse gases
produced from conventional diesel or fossil fuels. Lipases with excellent biochem-
ical and physiological properties are most commonly used to catalyze the trans-
esterification process for biodiesel production. Lipases obtained from microbes
such as bacteria and fungi produce 70%–95% ethanol and methanol. Biodiesel is
usually composed of fatty acid alkyl esters which are mono-alkyl esters of either
fatty acid methyl esters or fatty acid ethyl esters depending upon the alcohol (acyl
acceptor) being used in the reaction. Factors such as bioreactor type, acyl accep-
tor, temperature, and glycerol can affect the enzymatic transesterification reaction.
Recombinant enzymes such as recombinant lipases can be employed to obtain
higher percentage of biodiesel due to their high specificity and biocatalytic activity
for different substrates used for biodiesel production.
Keywords: Lipases, biodiesel, biocatalysis, biofuels, Novozyme, free fatty acids,
ethyl acceptors
*Corresponding author: habibullah@gcuf.edu.pk
Inamuddin, Mohd Imran Ahamed, Rajender Boddula, and Mashallah Rezakazemi (eds.) Biodiesel
Technology and Applications, (1–58) © 2021 Scrivener Publishing LLC
1
2 Biodiesel Technology and Applications
1.1 Introduction and Background

Biofuels are crucial for the conservation of our natural environment and
the climate. Biofuel such as bioethanol can be used for energy generation
purposes which are currently being produced by fossil fuels such as petrol,
diesel, and kerosene oil [1]. Being non-renewable energy sources, fossil fuels
will not only deplete from the planet earth but will also leave a long-term
impact on the globe both in terms of economy and climate change. Apart
from being limited natural fuel reserves, there are countless reasons available
that justify the need of natural and eco-friendly energy sources such as bio-
fuels. Transportation, power generation, and house hold appliances use fuels
directly or indirectly and for that purpose we are almost dependent on fossil
fuels [2]. If efficient and robust methods and technologies are not worked
out, we might come to a permanent stand still condition in the future when
all our natural fossil fuel reserves will be vanished. The use of fossil fuel pro-
duces gases such as carbon dioxide (CO2), carbon monoxide (CO), sulfur
oxides (SOx), and nitrogen oxides (NOx) which are unhealthy for human
beings causing health issues such as asthma, skin diseases, and even cancers
[3]. These by-products of fuel consumption affect not only human but also
animals and plants on a broader view. Plant production and growth rates are
highly effected by the changing environmental and climatic conditions due
to heavy use of fossil fuels and their derivatives such as plastics [4].
Vehicular CO2 emission in the past decade was 20%, and it is estimated
that by 2030, it will reach up to 80%. Liquide biofuels got prominence with
the automobile industry. Peanut oil was used to make biofuel, i.e., biodiesel by
Rudolph Diesel in 1898. Henri Ford who was the founder of Ford Company
an automobile industry was also convinced by the idea of using biofuels in
his automobile. During World War-II, Germany used biomass-based fuels for
their machines which is the evidence of its use back in 1940s. The utilization
of biofuels was presented, but after two major oil crises, first was in 1973 and
second in 1978, and brought back its importance to public again. Biofuels that
are produced using a large number of biomass sources are a sustainable solu-
tion for the environment and biosphere conservation. Being renewable energy
resources and eco-friendly to the environment and life on earth, these are
highly desirable products produced from renewable biomass substrates [5].
Currently, biofuels from various agricultural sources such as soybean oil,
rapeseed oil, recycled waste oils, and waste plant residues are being studied.
Depending on the feedstock type, processing technology and their devel-
opmental level, biofuels can be classified into first-, second-, and third-
generation biofuels. Biofuels produced directly from edible feedstock such
Biocatalytic Processes 3
as crops, sugars, and edible oil using conventional techniques are considered
as first-generation biofuels [6]. Non-edible feedstock such as waste crop res-
idues like lignocelluloses and waste vegetable oils are required to produce
second-generation biofuels which are comparatively economical and more
sustainable as there is no food versus fuel competition. Highly advanced
methods are used to produce second-generation biofuels which has certainly
less flaws and ultimately improved to get greater yield [7]. We are currently in
the phase of second-generation biofuels. Most of the processing techniques
for second-generation biofuel production are not available at commercial
level. One must think that the land dedicated for edible feedstock/crops will
be compromised if we start cultivating non-edible crops in that land. Marginal
lands can be used for the cultivation of grasses and other plants that are not
a food for human or nor a fodder for animals on a larger scale. These plants
or marginal grasses can be used for the production of second-generation bio-
fuels. There have been a lot of research investigations to produce biodiesel
using non-edible plant oils such as keranja oil, Jatropha curcas oil, tobacco oil,
Calophyllum inophyllum oil, and castor oil [8]. Jatropha is an effective source
of biodiesel production because of 30%–50% oil contents in its seeds [9]. The
actual precursors of most of the second-generation biodiesel production are
waste oils either in the form of waste cooking or industrial oils or animal fats.
The utilization of these waste materials as feed stock helps in managing and
disposing of waste material, which is one of the biggest problem for earth, for
the benefit of environment [10]. In order to comprehend different biofuels,
we can categorize them into four types which include biodiesel, bioalcohol
(biomethanol, bioethanol, biobutanol), biogas, and biohydrogen. The most
widely used biofuels are liquid biofuels such as biodiesel and bioethanol.
Biofuels can be blended with other petro-based fuels in order to manage and
enhance quality and quantity of fuel. Biofuel production includes chemical,
thermal, and enzymatic methods. Among all methods, the most effective way
to produce biofuels is through enzymes or biocatalysts [11]. Enzymes are
becoming the focus of research to produce biofuels because of their advan-
tages over other biofuel production techniques [12]. In this chapter, we dis-
cuss biodiesel production using biocatalytic processes and methods where
different microbial enzymes (obtained from microorganisms) are used.
1.2 Importance of Biodiesel Over Conventional

Diesel Fuel
Chemically, biodiesel is composed of fatty acid alkyl esters (FAAEs) which
are mono-alkyl esters of either fatty acid methyl esters (FAME) or fatty
acid ethyl esters (FAEE) depending upon the alcohol (acyl acceptor) being
used in the reaction [10]. Rudolf Diesel, the inventor of diesel engine, first
used biodiesel in 1900 but that was highly viscous so that engine could
not run effectively for a longer time [13]. Biodiesel is very suitable alter-
native to diesel fuel because of its remarkable properties and advantages,
i.e., biodiesel carries 4.5 times greater energy than fossil fuel [14] and sim-
ilar in chemical structure and energy content to conventional diesel [15].
It reduces approximately 85% carcinogenic compounds emission that is
why it is very less toxic than conventional diesel fuel, free of sulfur, free of
polycyclic aromatic hydrocarbons and metals, biodegradable, high cetane
number (CN), and flash point [16]. It has the potential to reduce pollutants
and emission of greenhouse gases [17] and is 66% more efficient lubri-
cating agent than petro-diesel, which enhances life and performance of
engine [18]. Blending of biodiesel with petro-diesel fuel that can affect
important properties of fuel such as flash point, CN, kinematic viscosity,
and lubricity is enhanced. It also decreases exhaust emissions and heat of
combustion [19]. The largest biodiesel producer is EU (European Union)
and biodiesel accounts 80% of the overall transport fuel in EU [20–22].
Biodiesel produced from different resources will have different composi-
tion and properties, but it must fulfill the standards and requirements of
international standards of American society for testing materials and EU
standards for biodiesel. Biodiesel has lots of applications such as it can be
used as a fuel for aviation purposes [20], for electricity production using
generators [21, 22] and in diesel fueled marine engines, because of its non-
toxic and biodegradable properties environmental impacts on engines can
be reduced. Alcohol type, quality of substrate that is to be converted, cata-
lyst used, temperature of the reaction, and alcohol-to-oil molar ratio deter-
mine the performance of biodiesel production [23–25].
1.3 Substrates for Biodiesel Production

Biodiesel feedstock accounts for 60%–80% of the total cost; therefore,
appropriate feedstock is required for economically valuable production
of biodiesel [26]. In order to obtain economically beneficial and sustain-
able biodiesel, feedstock must be easily available, cheap, and sustainable.
Feedstock is selected on the basis of biodiesel production that must be
compatible to chemical composition and properties of feedstock to be
used, percentage per dry biomass, agricultural potential, yield per hect-
are, and geographical region of that feedstock [27]. For example, soybean
oil, palm oil, coconut oil, and rapeseed oil are mainly used as feedstock in
US, tropical countries like Indonesia, coastal areas, and European coun-
tries, respectively. Cultivation and climate conditions of the feedstock pro-
duction area are also considered for its selection [28]. Depending on the
nature, there are two types of feedstock for biodiesel production. First is the
lipid raw material and second includes alcohol feedstock. Lipid sources can
be divided into three categories, i.e., oils derived from plant sources (edible
and non-edible oils), waste oils (waste cooking oils, industrial wastewater,
lard, yellow grease, and animal fats), and oils from oleaginous microorgan-
isms such as bacteria, fungi, and microalgae [29]. Properties of biodiesel
like cold filter plugging point and oxidation stability are determined from
the feedstock used for production. Feedstock properties like moisture con-
tent, impurities, content, and composition of free fatty acids (FFAs) affect
the performance of engine [27, 28]. Composition of fats and oils including
monoglycerides, diglycerides, and triglycerides are used for biodiesel pro-
duction. Utilization of edible plant oils as feedstock is an expensive way for
biodiesel production that leads to imbalance in food market and indus-
try. It is also associated with some environmental problems like disruption
of vital soil resources and deforestation due to mass propagation [29]. In
order to solve problems linked with edible plant oils, the best alternate is
the production of second-generation biodiesel which is produced by using
non-edible (inedible) feedstock which are more favorable than edible oils
due to reduction in cost and waste pollution, lower aromatic, sulfur con-
tents, and high calorific value [8]. Inedible oils involve inedible plant oils,
industrial waste, cooking oils, animal fats, and microalgal oils. Inedible oil
producing plants have certain remarkable features that make them favor-
able to use, for example, they can be managed to grow in arid and semi-arid
conditions and they do not require fertilizers and moisture for growth [24].
Repeated use of fried vegetable oils at high temperature leads to the
production of waste cooking oils. Moreover, chemical composition of
waste cooking oil is totally dependent on the oil from which it is derived.
Hydrogenation, oxidation, and polymerization are the main chemical
reactions that lead to production of very toxic and detrimental compounds
for consumption. Fatty acid content of these oils lies in the range of 0.5%
to 15% which is very much higher than refined oil having fatty acid con-
tent less than 0.5%. The waste cooking oil is known as yellow grease if the
fatty acid content is less than 15% and it is called low value brown grease
if the fatty acid content is higher than 15% [28]. Animal waste products
like lard, tallow, animal fat, poultry fat, fish oil, and pork fat are also very
effective feedstock for biodiesel production [30, 31]. Animal-based bio-
diesel is a good lubricating agent and has high percentage of saturated
fats which decreases sedimentation risk and low temperature fluidity.
Moreover, it increases oxidative stability and cold filter plugging point of

biodiesel which are the characteristics of good quality biodiesel. Utilizing
these waste materials is an effective solution to encounter waste disposal.
Apart from all these mentioned advantages of non-edible or waste oils,
there are also some shortcomings or disadvantages, for example, low oil
yield, higher carbon residue, unsaturated fatty acid content, and low vola-
tility [29]. In some cases, large plantation land for inedible oils is required
compared to edible ones, e.g., Pongamia pinnata and Jatropha has 2–50
folds less oil yield per hectare than palm oil so that is why they require
much area to meet the demand [31]. Because of the drawbacks associated
with second-generation biodiesel, scientists are looking for more efficient
methods for biodiesel production. Biodiesel production using oleaginous
microorganisms like bacteria, algae, microalgae, and fungi are considered
as the future of biodiesel production that can meet global biodiesel demand
for transportation fuels and other energy consuming applications [32].
Microbial oils are better than other plant oils because of their short life
cycle and rapid growth, less requirement of space, labor, and easier scaling
[33–35]. Microalgae as a feedstock is very effective because of its enormous
advantages like they have high oil yield, can grow in salty and waste waters,
use of non-arable land, and growth in 24 hours so multiple harvesting in a
year is possible. If we give land area for microalgal growth then according
to an estimate, only 2% of the US cropping land is enough for meeting
1/3 demand of US transportation fuels and less than 5% land is required
to completely replace all transportation fuels [36–38]. Moreover, dry algal
biomass can accumulate more than 80% oil without water and they have
a capacity to produce oil yield 250 times greater than soybean water free
oil [35]. Some examples of microalgae used for biodiesel production are
Botryococcus sp., Cylindrotheca sp., Schizochytrium sp., Chlorella sp., and
Nitzschia sp.
1.4 Methods in Biodiesel Production

There can be many ways for biodiesel production but esterification and
transesterification are the two most widely used methods. Esterification is
the reaction of FFAs and alcohol to make FAAEs and water is released, while
transesterification is the reaction of triglycerides or triacylglycerols (TAGs)
with alcohol to make FAAE and glycerol is produced as by-product [9].
Transesterification is slower than esterification process because of its multi-
ple steps or reactions. It is a three-step process to convert TAGs into FAAE.
In the first step, TAG reacts with one molecule of alcohol to produce one
molecule of FAAE and diacylglycerol (DAG). In second step, DAG further

reacts again with one molecule of alcohol to produce one molecule of FAAE
and monoacylglycerol and in the last step monoacylglycerol is converted
into one molecule of glycerol and FAAE after reacting with an alcohol mole-
cule. In each of these three steps, FAAEs are produced and in total one mol-
ecule of TAG and three molecules of alcohol are consumed to produce three
molecules of FAAE and one molecule of glycerol [6–10]. Transesterification
is a reversible reaction, and in order to make the reaction go forward to
produce more biodiesel, we have to supply alcohol in large excess so that the
reaction equilibrium shifts toward the product [36, 37].
1.5 Types of Catalysts Involved in Biodiesel

Production
Biodiesel production process is carried by either catalytic or non-catalytic
methods. Non-catalytic methods include use of alcohols or supercriti-
cal fluids or ionic liquids in the reaction system to produce biodiesel but
mostly catalytic methods have been used for last 2 or 3 decades because of
their advantages over non-catalytic methods [38]. Catalytic methods can
be categorized into chemical homogenous catalysts, solid heterogenous
catalysts, and biocatalysts.
1.5.1 Chemical Homogenous Catalysts

Chemical homogenous catalysts include combination of base and acid
catalysts. NaOH, KOH, and methoxides are the base catalysts while HCl
and H2SO4 are the acid catalysts [39]. Acid catalysts are mostly used to
overcome the problem of FFAs in the reaction system but the rate of trans
esterification by acid catalyst is slower than alkaline or base catalysts [8].
Chemical catalytic processes either alkaline or acid catalysis both have sev-
eral disadvantages. Alkaline catalysis provides high conversion of triacyl
glycerol into the alkyl esters in a very short time but it has many draw-
backs. Alkaline catalysis is very prone to FFA concentration (>2.5%) in the
reaction system because high FFA concentration results in saponification
reaction producing soaps and leads to loss in enzymatic activity and makes
difficult to separate transesterification by-product, i.e., glycerol from bio-
diesel. Hence, biodiesel yield decreases. Moreover, it needs high energy
requirement [40]. To counter FFA problem, acid catalysts are used, e.g.,
sulfuric acid but it also causes some technical problems regarding separa-
tion and purification of glycerol. Moreover, acid catalysis is a slow process
compared to alkaline process. Reactors, pipelines, and other equipment are

badly affected by acid catalysts because of their corrosive nature that can
increase the cost of biodiesel production [41].
1.5.2 Solid Heterogeneous Catalysts

Solid heterogeneous catalysts include acid heterogenous catalysts and base
heterogenous catalysts. Solid acid heterogenous catalysts include hetero-
polyacid catalysts (HPAs), mineral salts, acids, and cationic exchange res-
ins. Among these, titanium oxide, sulfonic ion exchange resin, tin oxide,
sulfonated carbon-based catalysts, zirconium oxide, zeolites, and sulfonic
modified mesostructured silica are the main acid heterogeneous catalysts.
Solid base heterogeneous catalysts have been categorized as mixed metal
oxides, supported alkaline earth metals, single metal oxides, and nano-
oxides. Among these, the most studied are magnesium oxide, calcium
oxide, and strontium oxide [44, 45].
1.5.3 Biocatalysts
Biocatalysts include enzymes especially lipases which are very popular in bio-
diesel production [43]. Enzymatic biodiesel production method diminishes
problems associated with alkali and acid catalyzed methods. Use of enzyme
catalysts has several economic and environmental advantages over chemi-
cal biodiesel production processes. Advantages of enzyme catalysis include
production of pure and high market value glycerol, minor, or no waste water
generation that is why treatment of waste water is not required, mild reaction
conditions are required, no soap formation because enzymes can esterify low
quality feedstock having high concentration of FFA that is why this method
is insensitive to feedstock concentration. Enzymatic biodiesel production is
simple so energy consumption is very low, enzymes can be reused because of
their easy separation from the reaction mixture, and overall chance of con-
tamination is lower than other transesterification methods [13].
1.6 Factors Affecting Enzymatic Transesterification

Reaction
There are a lot of factors effecting enzymatic transesterification reaction
such as source of enzyme, its type, preparation method, applying tech-
nique, its dosage, activity, and life time. Apart from these enzymes related
factors, there are also some other factors which affect transesterification
reaction, e.g., feedstock type and its quality, type of alcohol as acyl accep-
tor, reaction pH, presence or absence of solvent, type of solvent, reaction
temperature, alcohol-to-oil molar ratio [46].
1.6.1 Effect of Water in Enzyme Catalyzed Transesterification

Presence of water is not only required for chemically catalyzed biodiesel
production but also very much required for enzymatic biodiesel produc-
tion. It helps in maintaining enzyme structural confirmation and stabil-
ity so it directly affects activity of enzyme. Oil-water interface is required
for enzyme-substrate complex to proceed and water helps to increase this
interfacial area [44]. So, without water, transesterification is not possible
and absence of water can lead to permanent or temporal changes in protein
(enzyme) structure. If water content is minimal, then increase in water con-
centration moves the reaction equilibrium toward more hydrolysis. Thus, it
enhances reaction rate by providing greater stability to enzyme [45]. Excess
of water content also has some negative effects on the reaction as well as on
enzyme. Excess water content can be accumulated in the reaction medium
and within enzyme active site, that leads to decrease the reaction rate as
well as its alkyl ester yield [46]. So, concentration of water should be opti-
mally perfect in order to gain maximum benefit from it. Every enzyme has
its specific water content requirement, i.e., optimal water requirement, at
which that particular enzyme performs its best [47, 48]. Optimal water con-
tent not only provides great support, flexibility, and stability to the enzyme
but also maximizes transesterification yield by diluting methanol that has
an inhibitory effect on enzyme. Factors that determine optimal water con-
tent include feedstock and type of solvent used, enzyme, and its immo-
bilization technique used [48]. Chaudhary et al. [49] studied the effect of
water content in lipase catalyzed transesterification. At low water activity
(aw = 0.33), synthetic activity of enzyme was increased and at high water
activity (aw = 0.96) enzyme became more hydrolytically active. They tested
various enzymes/lipases at different water activity to check transesterifica-
tion rates. The lipase from Aspergillus niger was found more prominent to
give maximum transesterification rate of 0.341 mmolmin−1 mg−1 at aw = 0.75.
Measuring water content as weight percentage is a better choice and more
convenient to use than water activity (aw), measured by Karl-Fischer method
[50]. Maximum methyl ester yield was at water concentration of 10-15%
while increasing water content from 0% to 40% to study the effect of water
in conversion of salad oil into methyl ester. But after much increased water
concentration, methyl ester yield became very low. So, for maximum trans-
esterification yield, optimum water concentration is required.
1.6.2 Effect of Bioreactor

In order to maximize production and benefit of product we need to per-
form optimized laboratory experimental procedure at a large industrial
scale, so, bioreactors are used in this regard. But results should be equiva-
lent to laboratory procedure [45]. There are some complications like pro-
duction should be cost effective and in good quality. Carefully planned
methodologies and objectives should be designed for effectively large-scale
production. This also includes bioreactor parameters like fluid flow per-
formance and unexpected environmental variation. In case of industrial
transesterification process, the main hurdle is multiphasic nature of lipase
catalyzed synthesis and hydrolysis because this does not allow the bioreac-
tor equivalent to laboratory experiment. Many types of bioreactors such as
fluid beds, recirculation membrane reactors, expanding beds, static mixers,
batch stirred tank reactors (STRs), and packed bed reactors (PBRs) have
been used for enzymatic biodiesel production [51, 52]. One of the leading
differences between STRs and PBRs is presence of enzyme at specific loca-
tion in reactor, e.g., in STRs it is dispersed in the reaction mixture but in
PBRS it is fixed in a column. STRs are the simplest type of bioreactors con-
taining just reactor and propeller that stirs reaction mixture mechanically.
Batch operated STR need to be empty, clean, and again add reactants for
the reaction in order to start new batch process and this is main reason of
batch process to produce less yield of the product. Solution of this problem
is to use STRs with continuous mode. This does not require to remove
enzyme and ingredients to start another cycle. There is a filter attached at
the reactor outlet that preserves enzyme in the reactor [52]. PBRs can also
be used in both batch and continuous mode but later is more advantageous
because of its low labor cost, stable and automated controlled operating
conditions, high efficiency, protects enzyme from shearing stress, continu-
ous glycerol removal, and ease of maintenance [53–55]. Currently, most of
the bioreactors are used in batch mode with STRs but a lot of research has
been done on PBRs usage and its optimization for enzymatic biodiesel pro-
duction to find this PBR method is better than batch mode STR [56–59].
1.6.3 Effect of Acyl Acceptor on Enzymatic Production

of Biodiesel
Alcohols are mostly used as an acyl acceptor for biodiesel production. To
get maximum economical profit at industrial scale, acyl acceptor (alcohol)
should be cheap and readily available and that is why ethanol and especially
methanol are widely used for this purpose. Usually, three moles of alcohol
are required for each mole of oil and in order to keep the reaction moving
forward [56]. By increasing alcohol concentration, yield also increases but
up to a certain limit [57]. Methanol as an acyl acceptor is frequently used for
biodiesel production [58] because it is less expensive, has low chain length,
more volatile, and more reactive, and gives high yield than other alcohols
[55]. A lot of research has been done utilizing methanol as acyl acceptor to
convert various types of oils such as soybean oil, jatropha oil, and canola
oil, in the presence of free or immobilized lipase 96.4% yield of FAME was
obtained from microalgal oil using methanol as acyl acceptor in the pres-
ence of Candida rugosa lipase immobilized on bio-silica polymer [59, 60].
Different alcohols with different substrates may result in different yield so
alcohol-substrate combination should be kept in mind for maximum output.
Excess of methanol causes inhibitory effect in the reaction because it changes
the stability and configuration of biocatalyst/lipase that can leads to partial or
complete inactivation of lipase [60–62]. Moreover, it also causes hindrance
in separation of glycerol [61, 62]. Methanol inhibition was observed with
Novozym® 435 lipase in transesterification of waste oils [63], microalgae
oils, and various vegetable oils [64–67]. Inhibitory effect was also observed
with some other lipases such as lipases obtained from Rhizopus oryzae and
Burkholderia glumae [65]. Addition of alcohol in each step should be done
after considering type of substrate and enzyme to determine alcohol sub-
strate molar ratio [66]. This method of sequential addition of alcohol in reac-
tion system was first performed by [67]. 98% biodiesel yield was obtained
utilizing T. lanuginosus lipase to convert soybean oil using stepwise addition
of methanol [68]. Inhibition of lipases such as C. rugosa lipase, P. cepacia
lipase, R. oryzae lipase, and P. fluorescens lipase was prevented using step-
wise addition of methanol and 90% yield was also obtained by converting
waste cooking oil into biodiesel [69]. Methanolysis of olive oil increases by
34% using stepwise addition of methanol compared to batch methanolysis
[70]. Transesterification of waste cooking oil using Novozym 435 was also
reported to yield 93% and 96% conversion for continuous and batch process
and lipase did not lose its activity even after 20 cycles [50]. Three-step addi-
tion of methanol resulted in 97% conversion of plant oil with 0.25- to 0.4-h
intervals. But this method of stepwise addition requires low level mainte-
nance of methanol concentration so it cannot be effectively used for indus-
trial scale. Alcohols as an acyl acceptor other than methanol include high
chain primary alcohols, secondary, branched, and linear chain alcohols such
as ethanol, isopropanol, t-butanol, and octanol [71].
Choice and selection of appropriate alcohol is important as it can
influence some biodiesel properties like lubricity and cold flow proper-
ties [75, 76]. Moreover, high chain alcohols cause less lipase inhibition
and produce high yield than methanol because lipase show more affin-
ity toward higher chain alcohols than lower chain [12]. The most widely
used alcohol as an acyl acceptor after methanol is ethanol as it is less
inhibitory, less toxic, and derived from renewable resources [73] unlike
methanol which is derived from coal and natural gas. There is minor
difference between characteristics of fuels obtained after methanol and
ethanol, i.e., FAME and FAEE, respectively. As FAEE has large viscosity
and lower pour and cloud points [74, 75]. Hernandez-Martin and Otero
[76] showed that, Novozym 435 catalyzed transesterification of sun-
flower oil, that was performed using methanol and ethanol separately
to check which acyl acceptor would perform better. Methanol-mediated
transesterification showed more lipase inhibition than ethanol contain-
ing reaction. Moreover, ethanol transesterification reaction was faster
than methanol reaction. Acyl acceptors other than alcohols can also be
used as an alternative such as methyl acetate, ethyl acetate, and dimethyl
carbonate (DMC). Methyl acetate was utilized as an acyl acceptor for
transesterification of soybean oil catalyzed by Novozym 435. In addition,
92% methyl ester yield was obtained [77]. Similarly, >90% ethyl ester
yield was obtained when utilizing ethyl acetate as an acyl acceptor for
transesterification catalyzed by Novozym 435 [78].
Use of DMC resulted in over 90% yield even after 10 times reuse of
Novozym 435 lipase to convert Chorella sp. KR-1–derived triglyceride
[79]. But use of methyl acetate and ethyl acetate is cost expensive and
also make the product difficult to separate. Another strategy can be used
to reduce methanol inhibition problem, i.e., use of solvents in the reac-
tion mixture [80]. Use of solvents is beneficial for various reasons such
as it increases solubility of alcohol and glycerol that results in preven-
tion of lipase denaturation [81]. It increases the rate of reaction because
it improves mass transfer rate. Use of solvents do not allow to form new
separate phase that hinders enzyme activity because it dissolves most part
of alcohol that makes a separate phase if remained undissolved. Moreover,
it reduces viscosity and stabilizes lipase [45, 55]. Enzyme stabilization is
associated with the presence of water molecules and their activity sur-
rounding the lipase structure. So, use of polar, less hydrophobic solvents
is not a good idea because that can lead to distortion of enzyme con-
firmation [82]. A higher yield of FAME was obtained from microalgae
lipids catalyzed by intracellular lipase when non-polar n-hexane solvent
was used as compared to polar tert-butanol solvent [83]. Organic solvents
such as hexane, petroleum ether, tert-butanol, n-heptane, and ionic liq-
uids are widely used for lipase catalyzed transesterification purpose [88].
Sometimes, it also happens that use of solvents becomes necessary in
transesterification reaction if short chain alcohols are being used as an

acyl acceptor in order to completely dissolve alcohol and produce max-
imum output but for the same reaction conditions solvent-free reaction
system can be used if higher chain alcohols are used as an acyl acceptor.
Iso et al. [85], immobilized P. fluorescence lipase catalyzed transesterifica-
tion was performed using methanol and ethanol as an acyl acceptor. They
also provided 1,4-dioxane solvent to the reaction to carry out effective
transesterification reaction. But when they used propanol and butanol as
an acyl acceptor, they did not provide any solvent to the reaction because
addition of solvents was not necessary required for transesterification.
Without solvent reaction worked and appropriate result was obtained.
Similar type of findings was also observed in another experiment that
in which hexane as solvent was used for methanolysis of various oils or
substrates like rapeseed oil, soybean oil, recycled restaurant grease, and
tallow. That reaction was catalyzed by C. antartica lipase (SP 435) and
M. miehei lipase (lipozyme IM 60) [53]. Solvents stabilized the lipase activity
shown by Li et al. [86], where lipase AK did not loss its activity. 1,4-dioxane
was used as solvent and gave higher yields. Presence of t-butanol as sol-
vent also resulted in the improvement of methyl ester yield [84].
Effects of various solvents like benzene, tetrahydrofuran, chloroform,
and 1-4 dioxane were investigated using different enzymes such as P. cepa-
cia (Lipase PS), C. rugosa (Lipase AY), M. javanicus (Lipase M), P. fluo-
rescens (Lipase AK), and R. niveus (Newlase F) by [85]. Use of solvents
definitely increases the reaction rate and solubility of alcohol which is ben-
eficial but it is not economical and environment friendly because to sepa-
rate organic solvent from reaction mixture a solvent recovery unit is also
required that increases the cost of recovery. Its flammability and toxicity
is another concerning factor [87]. Choice of lipase according to alcohol
is also important as some lipases show more resistance toward different
alcohols. Yang et al. [88] found that Photobaterium lipolyticum lipase was
more tolerant to methanol inhibition than C. antartica lipase B (Novozym
435) when transesterification was performed using one step methanol
addition. Similarly, Pseudomonas lipases were found to be more alcohol
tolerant compared to lipases from R. miehei and T. lanuginosus. That is why
pseudomonas lipases have higher methanol-to-oil molar ratio. Out of nine
lipases, only Pseudomonas cepacia lipase showed high ester yield from soy-
bean oil with 8.2:1 methanol-to-oil molar ratio [93, 94]. A recent and novel
approach of countering methanol inhibition is addition of silica gel in the
reaction system. Silica gel absorbs methanol and keeps its concentration
level below to prevent lipase inhibition. But presence of silica gel makes
separation of products difficult.
1.6.4 Effect of Temperature on Enzymatic Biodiesel Production

Use of enzyme in any chemical reaction makes the reaction less energy
intensive, but, like every other chemical reaction, increase in temperature
enhances reaction speed and rate. Similarly, in case of enzymatic biodiesel
production, increase in temperature increases enzyme activity, reaction
speed, its rate, and production yield [45]. When Lipozyme TL IM lipase
was used to transesterify crude palm oil using methanol then the resulting
FAME yield was 96.15% and 85.86% at 40°C and 30°C, respectively [95,
96]. But this effect is limited to certain extent because beyond enzyme opti-
mum temperature, enzyme structure becomes unstable and that leads to
enzyme denaturation and reaction becomes slower and yield also decreases.
Novozym 435 catalyzed biodiesel production from microalgal lipids, there
was 19% decrease in product yield when temperature went from 45°C to
55°C, i.e., higher than optimum temperature [97, 98]. Enzyme tempera-
ture should remain below the boiling point of alcohols being used in the
reaction system to avoid evaporation of alcohol. In case of methanol and
ethanol-mediated transesterification, reaction temperature is 65°C and
78°C, respectively [8]. Free bacterial lipases are considered thermally sta-
ble but if they get immobilized thermal stability increases [45]. Optimum
enzyme temperature is influenced by lipase thermal stability, type of sol-
vent, alcohol-to-oil molar ratio, and lipase immobilization. Every enzyme
has different optimum temperature depending on the source and type
of enzyme. Normally, lipases have optimum temperature range that is
20°C–70°C. Optimum temperature for C.antartica lipase is 40°C [99, 100].
1.6.5 Effect of Glycerol on Enzymatic Biodiesel Production

Glycerol is another product obtained along with TAG in transesterification
reaction for biodiesel production when alcohols are used as an acyl acceptor.
Like methanol inhibition effect, glycerol also hinders enzymatic transester-
ification. Production of glycerol cause reversal of reaction equilibrium and
thus opposes biodiesel production. Glycerol insolubility in the reaction sys-
tem makes it to accumulate in the system and increases the viscosity of the
reaction mixture. Not only it just increases viscosity but because of its hydro-
philic nature, it surrounds the lipase and hence prevents substrate to interact
with enzyme and binding at enzyme catalytic site. This effect is more promi-
nent when enzyme is in immobilized state. These things all together impacts
negatively on the transesterification reaction [93]. According to a research
study, rapeseed oil was transesterified using ethanol as an acyl acceptor with
different immobilized enzymes such as Novozym 435, Lipozyme TL HC
(immobilized on polymeric resin) and Lipozyme TL IM (immobilized on

silica). Among all reactions, glycerol became more accumulated and hin-
dered reaction when silica was used for immobilization purpose because it
had large number of micropores that helped in accumulation [94].
A lot of methods have been devised to overcome and tolerate glycerol
inhibition problem such as continuous removal of glycerol, use of solvents,
and use of acyl acceptors other than alcohols. Biodiesel production in PBR
is effective for continuous production and also to tolerate glycerol inhibi-
tion because it allows continuous removal of glycerol from it [95]. Bélafi-
Bakó [96] showed that 97% conversion yield was obtained by methanolysis
due to continuous removal of glycerol. Use of solvents is another strategy
to resolve glycerol inhibition problem. Solvents, for example, tert-butanol
and ionic liquids dissolve glycerol and thus reduce the glycerol inhibition
problem. Moreover, lipases also perform better in the presence of solvents
[97]. Azócar et al. [4] inferred that inhibition effect was eliminated when
tert-butanol was used as solvent to convert soybean oil into biodiesel pro-
duction in a continuous way because it is an excellent solvent for methanol
and glycerol to dissolve in it and, thus, reduces inhibitory effects of both
methanol and glycerol. These methods have shown promising work but
these are not as good for industrial scale production.
There is another novel method in which instead of alcohols other
compounds like methyl acetate, ethyl acetate, and DMC are in use. Use
of methyl acetate or ethyl acetate does not produce glycerol as a product
along with the main product instead it leads to produce triacetylglyc-
erol that does not inhibit any enzymatic or reaction activity and further
downstream processes are not halted [98]. According to Zhang et al. [99],
transesterification of palm oil was done using Novozyme 435 as catalyst
and DMC as acyl acceptor with reaction conditions were 10:1 DMC to oil
ratio, 55°C reaction temperature and 20% lipase in a solvent-free system.
In addition, 90.5% conversion yield, i.e., FAME was obtained, and after
eight reaction cycles, no reduction in enzyme activity and loss of yield was
observed. It was just because glycerol was not produced instead glycerol
dicarbonate was formed because of DMC as acyl acceptor for the reaction.
After discussing all negativity about glycerol, it seems glycerol does not
have any positive effect but it’s not true. Glycerol if purified absolutely from
the transesterification, as mostly huge biodiesel producing companies do,
has vast number of uses in diverse industrial fields. Moreover, 99.7% puri-
fied glycerol can be used as a raw material for various types of fields such as
paints, toiletries, animal feed, emulsifiers, pharmaceuticals, textiles, drugs,
tobacco, cosmetics, toothpaste, leather, plasticizers, paper, food, and for
different chemicals production [100, 101].
1.6.6 Effect of Solvent on Biodiesel Production

One of the major problems in enzymatic biodiesel production is enzyme
inhibition by short chain alcohols such as methanol and ethanol that are
used in the reaction. These alcohols’ insolubility in the reaction system
denatures the enzyme and hence reduces yield of biodiesel. So, solvent
application plays its role in this regard. Organic solvents are used to solubi-
lize these excessive alcohols so that enzyme denaturation can be prevented.
Hence, it stabilizes the enzyme. Solubility of oils and alcohols become
increased due to presence of organic solvents, this provides the required
environment for substrate to interact with enzyme at its active site. Organic
solvents also reduce viscosity of the reaction mixture and enhances mass
transfer toward the enzyme that leads to improved reaction rate [101].
Organic solvents also eliminate the need of stepwise addition of alcohol.
All these things in combination increase production of biodiesel. Organic sol-
vents that are commonly used include tert-butanol, petroleum ether, hexane,
and n-heptane [102]. Some other organic solvents that are used are 2-butanol,
cyclohexane, isooctane, acetone, 1,4-dioxane, and chloroform. While consid-
ering nature of organic solvents, hydrophobic organic solvents are majorly
used. Hydrophobicity of the organic solvents helps in accumulating water
molecules around enzyme which is important for enzyme structural stability
[103]. Polar or hydrophilic solvents work opposite to hydrophobic organic
solvents by playing role in distortion of enzymatic structure. But solvent with
little polarity can be beneficial to dissolve oil and alcohol. For example, hydro-
philic 1,4-dioxane and tert-butanol have produced some good results by pro-
ducing high enzymatic transesterification yield [104]. Tert-butanol, having
moderate polarity, eliminates glycerol and methanol inhibition problem for
enzyme because it can dissolve both in itself. This makes the enzyme more
stable and active and then ultimately produce better reaction yield [105]. Tert-
butanol is the most common solvent that proved its effectiveness in various
cases. According to Royon et al. [84], cottonseed oil was transesterified in the
presence of Candida antartica lipase. Methanol was found to be the cause of
enzyme inhibition in the reaction but when tert-butanol was used as solvent,
reaction yield goes up to 97% with minimal enzyme inhibition. Similarly,
in another research experiment, tert-butanol was tested for its effectiveness
when rapeseed oil was used as substrate for biodiesel production. In sol-
vent-free system, methyl ester yield was 10% but after utilizing tert-butanol
yield was 75%. But under optimum conditions having Lipozyme TL IM and
Novozyme 435 both in the reaction system, biodiesel yield reached 95% and
the reaction was so stable that enzymes did not lose their activity even after
200 cycles. Reaction was favored and well supported by tert-butanol [86].
Use of solvents provide many benefits but they also come with some
disadvantages such as organic solvents do not completely dissolve glycerol,
by-product of the reaction, that causes the enzyme to lose its activity and
become unstable. Use of solvents also make the process very costly because
there is a need of extra purification step to separate out solvent and product
from the reaction mixture. Organic solvents are mostly toxic and highly
flammable so there are also environmental and health concerns while
using them [11]. In order to tackle problems of conventional organic sol-
vents, researchers have suggested some alternatives. Diesel oil was found
to be an interesting alternative but the most recent, beneficial, and popular
alternatives are super critical carbon dioxide (SC-CO2) and ionic liquids
(ILs). Researchers have also confirmed the positive effect of using SC-CO2
and ILs in the enzymatic transesterification [106, 107].
1.7 Lipases as Biocatalysts for Biodiesel Production

Transesterification of oils for biodiesel production is done using either
chemical or enzymatic catalyst [108]. An enzymatic catalyst is used at first
place due to their normal reaction conditions, reusability, easy products
separation, and production of high-quality product. There is less energy
consumption in enzyme catalysis as it occurs at a low temperature as
compared to chemical catalysis requiring high energy consumption [109,
110]. Further, enzymatic catalysis is environment-friendly as there is no
wastewater production and produces pure biodiesel as compared to chem-
ical catalysis [107]. Among enzymatic catalysts, lipase with excellent bio-
chemical and physiological properties is most commonly used to catalyze
the transesterification process. Lipases play their role in several industrial
processes like alcoholysis, acidolysis, amynolysis, and hydrolysis reactions
but their leading role in biodiesel production is considered very important
[108–111]. The use of lipase in biodiesel production is proved to be bene-
ficial due to its characteristics like high efficiency, convert FFAs completely
into methyl/ethyl esters, reaction specificity, require low temperature, min-
imum energy consumption, and fewer side products [109]. Lipases belong
to class “hydrolases” as they carry out hydrolyses of triglycerides produc-
ing glycerol and fatty acids from it in an oil-water interface [110]. A general
reaction for biodiesel production using lipase is as follows:
lipase
Triglycerides + 3 Alcohol 3 Fatty acid methyl ester + Glycerol
(biodiesel) (by-product)
Lipases work on specific substrates and carry out catalysis of hetero-

geneous reactions in water-soluble as well as insoluble systems. Further,
lipases have the properties like chemo-specificity, region-specificity, and
stereo-specificity [111]. When classification is made based on region-
specificity, there come three classes of lipases: 1) non-specific lipases, 2) 1,3-
specific lipases, and 3) fatty acid-specific lipases. Non-specific lipases have
ability to attach with all the possible positions of triglycerides to give FFAs
and glycerol. The intermediates of the reaction, diglycerides, and mono-
glycerides do not accumulate in the reaction as they are instantly hydro-
lysed into fatty acids and glycerol [112]. 1,3-specific lipases are specific for
the 1 and 3 positions of triglycerides and remove fatty acids from these
positions. 1,3-specific lipases carry out the conversion of triglyceride to
diglycerides much faster than diglyceride to monoglyceride [113]. Fatty
acid-specific lipases carry out hydrolysis of a specific type of esters which
have double bonded long chains of fatty acids in cis position between C-9
and C-1. Hydrolysis of esters with unsaturated fatty acids occur slowly and
such class of lipases is not much common [114]. All the hydrolytic enzymes
including lipases have common folding pattern involve in a hydrolytic
activity called α/β hydrolase fold which is made up of a β sheet of eight
strands (one of which is antiparallel while remaining seven strands are
parallel) connected by α helices. Histidine residue, catalytic acid residue
and Nucleophilic residue are present in α/β hydrolase fold. Pentapeptide
sequence (Gly-X-Ser-X-Gly) which is a highly conserved in most of the
lipases involved in the construction of ‘nucleophilic elbow’ which is a typ-
ical β-turn-α motif having active nucleophilic serine residue between a β
strand and an α-helix. Catalytic triad made up of amino acids like his-
tidine, serine, and aspartic acid or glutamic acid build the active site of
lipases. The same catalytic triad is seen in serine proteases predicting com-
mon catalytic mechanism in them. Amphiphilic α helix peptide sequence
forms a lid or flap which covers the active site of lipase and has a struc-
tural variability depending upon the lipase source organism. Changes in
the structure of the lid are responsible for the activation/inactivation of
lipases [114]. Changes in the conformation of lipase structure as well as the
quality and quantity of interface being used in the reaction are responsible
for the activation of lipase. When the lipase enzyme meets the oil/water
interface there occur some changes in lipase structure that results in its
activation. For the activation of lipase first, the lid opens to uncover the
active site of lipase upon its contact with the ordered interface [115]. Due
to this restructuring of lipase, electrophilic region is created around serine
residue present in active site, lid hydrophilic side which was exposed in
native form now partly buried inside the polar cavity and hydrophobic side
of lid completely exposed, thus creating a non-polar surface around the

active site for efficient attachment of lipid interface with it [115].
1.7.1 Mechanisms of Lipase Action

Lipases interact with ester bonds of their substrate like acylglycerols to
catalyze the reactions of hydrolysis, synthesis, and transesterification.
Triglycerides, which are insoluble and long chained fatty acids, are precisely
catalyzed by lipases [113]. Lipase carries out triglyceride oil transesterifica-
tion with methanol in three reversible steps with the first step for conversion
of triglycerides to diglycerides followed by the second step of diglycerides to
monoglycerides conversion, and finally, monoglycerides convert into glyc-
erol molecules. Here, each conversion step produces one FAME molecule;
hence, a total of three FAME molecule are produced from one triglycer-
ide [116]. Two models are mainly under discussion to describe the kinet-
ics mechanism for esterification reactions, Michaelis-Menten kinetics and
Ping Pong Bi Bi model. Lipase catalyzed esterification mainly elaborated by
Ping Pong Bi Bi mechanism which is a bi-substrate reaction that releases
two products. It involves following steps: 1) acyl-donor donate their acyl
group to the enzyme resulting in the formation of acyl-enzyme complex,
2) release of the water molecule as a product, 3) binding of acyl acceptor
with the enzyme complex, and 4) release of ester [117, 118]. Many research-
ers made some modifications in this model depending upon inhibiting
factors [118]. The catalytic activity begins with the transient tetrahedral
intermediate formation with a negatively charged carbonyl oxygen atom.
The reaction between the hydroxyl group oxygen present in nucleophilic
serine residue of lipase enzyme and activated carbonyl carbon of the sub-
strate involved building this transient tetrahedral intermediate. The inter-
mediate thus formed is stabilized by its interaction with two peptide NH
groups. After that nucleophilic hydroxyl group of water react with the car-
bonyl carbon of acyl-enzyme complex resulting in the formation of acyl
product and enzyme is released for further catalysis [119].
1.7.2 Efficient Lipase Sources for Biodiesel Producing

Biocatalyst
Lipases can be obtained from plants, animals, and microorganisms and
based on that lipases can be classified on it as plant, animal, and micro-
bial lipases depending on their origin respectively. Lipases from different
sources with different structure has different properties and catalytic activ-
ities. We can use this to counter lipase problems in biodiesel production
like lipase cost and methanol inhibition. We need to optimize reaction

conditions according to chosen substrate and lipase from specific source
[107]. Microbial lipases are widely used at industrial and commercial
level as biocatalysts for biodiesel production because microbial lipases are
more stable and can be produced in bulk amount from microorganism
[23]. Microbial lipases can be manipulated genetically with ease, seasonal
changes have nothing to do with lipase production, and rapid growth of
microbes makes them the ideal candidates as lipase source [120, 121]. Use
of microbial lipase is increasing day after day and currently 5% of the world
enzyme market is being shared with microbial lipases [121]. Microbial
lipase weighs around 30–50 kDa and their optimum pH to work at is 7.5.
Based on temperature tolerance, microbial lipases can be categorized as
mesophilic and thermophilic lipases. Mesophilic lipases normally work at
35°C–50°C and they become denature above 70°C while thermophilic or
thermostable lipases normally work at 60°C–80°C, but some also work at
100°C under specific conditions [122, 123]. According to Hotta et al. [123],
lipases from Pyrobaculum calidifonti (hyperthermophilic archae) showed
its activity at 90°C. Similarly, thermostable lipases from Caldanaerobacter
subterraneus and Thermoanaerobacter thermohydrosulfuricus (highly ther-
mophilic bacteria) performed well in the range of 40°C–90°C. They not
only performed well at high temperatures but also were resistant toward
organics solvents [124]. Lipase producing microorganisms can be iso-
lated from soil, waste water, marine water, and industrial wastes. Isolates
of Mucor, Sclerotina, Candida, and Aspergillus strains have been reported,
which were isolated from soil. Similarly, various strains of microbes such
as P. alcaligenes, Bacillus acidophilus, Enterobacter intermedium, P. flu-
orescens, and Geotrichum asteroids are also reported as lipase producing
strains when isolated from vegetable oil processing plants [141]. Screening
of microbes is done by checking their lipolytic. Generally, lipases are
screened using batch cultures having agar as substrate but this is time con-
suming. So, the two mostly used methods for lipase production are solid
state fermentation and using submerged culture [142]. Purified form of
lipases is used in biochemical reaction to get maximum benefit from it, so
purification is required. Purification of lipase requires several techniques
including ammonium sulfate precipitation or ultrafiltration and after that
more sensitive and advanced techniques are utilized like gel filtration, ion
exchange chromatography, and affinity chromatography [143]. Moreover,
some other novel techniques can also be applied to purify lipases such as
immunopurification, column chromatography, hydrophobic interaction
chromatography, and membrane process. Generally, the strategy used for
purification lipases starts with the removal of lipase producing cells from
their growth culture to get extracellular lipases after fermentation. Then,

the extract without cells is concentrated by organic solvents, ultrafiltration,
and precipitation using ammonium sulfate. Ammonium sulfate precipi-
tation is used in the first stages of purification and it crudely separate out
things from mixture. After that advanced techniques of chromatography
are used to finely purify lipases [144]. According to Javed et al. [112],
diverse data from various research work suggested that purification had
been done from 2.4 to 500 folds with an increase in yield from 10.3% to
36%. Effective production and purification strategies of lipases are being
designed to get maximum yield at a very small expense. Among microbial
lipases, the most commonly used lipase sources are bacteria, fungi, yeast,
and algae; see Table 1.1 for some bacterial lipases and Table 1.2. for fungal
lipases that are used for biodiesel production [46]. Some of the commer-
cially available enzymes for biodiesel production are enlisted in Table 1.3.
1.8 Comparative Analysis of Intracellular and

Extracellular Lipases for Biodiesel Production
Transesterification reaction for biodiesel production is done with both
extracellular and intracellular lipases. Preference for their use is depen-
dent upon either we want simple upstream processes as in case of intra-
cellular lipase or high enzymatic conversion as in case of extracellular
lipase. But either we use intracellular lipase or extracellular lipase there is
no need for some downstream processes including separation and recy-
cling. Further, both immobilized lipase (extracellular) and immobilized
whole-cell lipase (intracellular) are proved to have highly efficient when
compared with free lipase used for transesterification [165]. Some exper-
imental studies for both intracellular and extracellular lipases are given
in Table 1.5. Intracellular lipases are the enzymes present inside the cells
or linked to the walls of cells producing it known as whole-cell biocat-
alysts. They are not purified or separated from their cells and used as a
whole-cell for transesterification (whole-cell biocatalyst) or immobilized
(whole-cell immobilization) [55]. Rhizopus and Aspergillus which are fila-
mentous fungi are most widely used as whole-cell biocatalyst for transes-
terification process [166]. As the main issue related to biodiesel production
at large scale is cost and the use of intracellular lipase for transesterifica-
tion resolves this problem like the use of intracellular lipase is considered
cost-effective because of the elimination of costly processes of lipase isola-
tion and purification before immobilization which are required in case of
extracellular lipase [167]. Intracellular lipase producing cells or whole-cell
Table 1.1 Some of the commonly used bacterial lipases for biodiesel production.
Acyl
Enzyme Immobilized on Substrate acceptor Yield Reference
Burkholderia Hydrophobic Jatropha oil Methanol 95% [125]

cepacia silica monolith
Lipase
Hybrid matrix of Jatropha Ethanol 100% [126]

alginate and curcas L.
κ-carrageenan oil
κ-carrageenan Palm oil Methanol 100% [127]
Modified Jatropha oil Methanol 94% [128]

attapulgite
SiO2-PVA Babassu oil Ethanol 100 % [129, 130]
SiO2-PVA Babassu oil Ethanol 100% [131]
Nb2O5 Babassu oil Ethanol 74.1% [132]
Pseudomonas Epoxy-acrylic Waste Ethanol 46–47% [133]

cepacia resin vegetable
Lipase oil
Phyllosilicate sol- restaurant Methanol 98% [134]

gel matrix grease and
Ethanol
Fe3O4 nanoparticle Soybean oil Methanol >88% [135]

biocomposite
Accurel Madhuca Ethanol 96% [136]

indica
Protein-coated Soybean oil Ethanol 98.93% [137]

microcrystals
Celite Jatropha oil Ethanol 98% [89]
Pseudomonas Octyl-silica resin Babassu oil Ethanol 97.5% [85]

fluorescence
Hydrophobic Soybean oil Methanol 65% [138]
sol-gel
Porous kaolinite Triolein Methanol 90% [139]

particles and
Ethanol
Asymmetric Triolein Methanol 80% [140]

membrane
Table 1.2 Some of the commonly used fungal lipases for biodiesel production.
Acyl
Enzyme Immobilized on Substrate acceptor Yield Reference
Candida Activated textile Waste cooking Methanol 91.08% [157]
antartica cloth oil
Lipase
Polyurethane foam Soybean oil Ethanol 81% [158]
Acrylic resin Sunflower oil Ethyl acetate 92.7% [78]
Soybean oil Methanol 83.31% [95]
Candida Microporous bio Scenedesmus Methanol 96.4% [59]
rugosa silica-polymer quadricauda
Lipase microalgal
oil
Poly(styrene- Soybean oil Methanol 86% [159]
methacrylic
acid)
microsphere
within an activated Palm oil Methanol 70% [160]
carbon as
support
Thermomyces Olive pomace Pomace oil Methanol 93% [161]
lanuginosus
Phyllosilicate sol-gel Grease Ethanol 80-90% [162]
Lipase
matrix
Mesoporous poly- Oleic acid Methanol 90% [163]
hydroxybutyrate and
particles (PHB) Ethanol
Toyopearl Babassu oil Ethanol 86.6% [164]
AF-amino-650M
resin
biocatalysts are directly employed for immobilization without separation

and purification steps for lipase enzymes [55]. Porous biomass support
particles (BSPs) are mostly used for whole-cell immobilization. BSPs was
developed by Atkinson et al. [168] and used by many scientists and each
scientist provide an efficient way of immobilization on it giving out a high
yield of biodiesel. A study to check the lipolytic activities of Bacillus species
using intracellular as well as extracellular lipase showed higher intracellu-
lar lipase activity than extracellular lipase activity [169]. Reported whole-
cell biocatalysts are Aspergillus oryzae, Burkholderia cepacia, filamentous
fungus Rhizopus chinensis, R. oryzae, and Enterococcus faecium [170–174].
Aspergillus oryzae used as whole-cell biocatalyst exhibited 98.1% relative
Table 1.3 Some examples commercial lipases commonly used for biodiesel
production.
Reaction
Enzyme Substrate Acyl acceptor yield Reference
Novozyme Chlorella sp. Dimethyl carbonate 90% [145]
435 KR-1 and methanol
mixture
Sunflower oil Methanol, Absolute >90% [146]
ethanol,
1-propanol
Oleic acid Ethanol, n-propanol, >90% [147]
and n-butanol
Crude Methanol 94% [148]
soybean oil
Soybean oil Ethyl acetate 63.3 % [149]
Soybean oil Ethanol [150]
Lipozyme Soybean oil Methanol >90% [151]
TL IM
Crude palm Methanol 96.15% [90]
oil
Waste Methanol 92.8% [152]
cooking oil
Palm oil Oleyl alcohol 79.54% [153]
Corn oil Methanol 92% [154]
Lipozyme Crude Monoacylglycerol 90% [155]
RM IM rapeseed
oil
Sunflower oil Methanol >80% [82]
Castor oil Ethanol 98% [3]
Soybean oil Ethanol >88% [156]
deodorizer
distillate
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Biodiesel Technology and Applications Inamuddin Full Chapter

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Biodiesel Technology and Applications

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Library of Congress Cataloging-in-Publication Data

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1.11 Recent Strategies to Improve Biodiesel Production 31

3.4 Biocatalysts 115

5.3.1 Feedstocks Popular in India 169

6.1 Introduction 196

8 Biodiesel Production Technologies 241

9.2.4.4 Variables Affecting Transesterification

11.5.3 Tall Oil 318

11.9.1.3 Elimination of Unsaponiﬁables 333

13.3.1 Homogeneous Catalyst 364

15.3 Waste Animal Fats 404

17.4.1 The Choice of Transesterification Catalysts 454

substrates for biodiesel production including their percentage yields are

intensification techniques to manage mass transfer restrictions of oil and

Chapter 16 provides detailed information on the fabrication of biodiesel

Inamuddin, Mohd Imran Ahamed, Rajender Boddula

College of Chemistry, Chemical Engineering and Biotechnology,

Donghua University, China

*Corresponding author: habibullah@gcuf.edu.pk

1.1 Introduction and Background

1.2 Importance of Biodiesel Over Conventional

1.3 Substrates for Biodiesel Production

Moreover, it increases oxidative stability and cold filter plugging point of

1.4 Methods in Biodiesel Production

molecule of FAAE and diacylglycerol (DAG). In second step, DAG further

1.5 Types of Catalysts Involved in Biodiesel

1.5.1 Chemical Homogenous Catalysts

compared to alkaline process. Reactors, pipelines, and other equipment are

1.5.2 Solid Heterogeneous Catalysts

1.6 Factors Affecting Enzymatic Transesterification

1.6.1 Effect of Water in Enzyme Catalyzed Transesterification

1.6.2 Effect of Bioreactor

1.6.3 Effect of Acyl Acceptor on Enzymatic Production

transesterification reaction if short chain alcohols are being used as an

1.6.4 Effect of Temperature on Enzymatic Biodiesel Production

1.6.5 Effect of Glycerol on Enzymatic Biodiesel Production

(immobilized on polymeric resin) and Lipozyme TL IM (immobilized on

1.6.6 Effect of Solvent on Biodiesel Production

1.7 Lipases as Biocatalysts for Biodiesel Production

Lipases work on specific substrates and carry out catalysis of hetero-

of lid completely exposed, thus creating a non-polar surface around the

1.7.1 Mechanisms of Lipase Action

1.7.2 Efficient Lipase Sources for Biodiesel Producing

like lipase cost and methanol inhibition. We need to optimize reaction

their growth culture to get extracellular lipases after fermentation. Then,

1.8 Comparative Analysis of Intracellular and

Burkholderia Hydrophobic Jatropha oil Methanol 95% [125]

Hybrid matrix of Jatropha Ethanol 100% [126]

κ-carrageenan Palm oil Methanol 100% [127]

Modified Jatropha oil Methanol 94% [128]

SiO2-PVA Babassu oil Ethanol 100 % [129, 130]

SiO2-PVA Babassu oil Ethanol 100% [131]

Nb2O5 Babassu oil Ethanol 74.1% [132]

Pseudomonas Epoxy-acrylic Waste Ethanol 46–47% [133]

Phyllosilicate sol- restaurant Methanol 98% [134]

Fe3O4 nanoparticle Soybean oil Methanol >88% [135]

Accurel Madhuca Ethanol 96% [136]

Protein-coated Soybean oil Ethanol 98.93% [137]