Cover
Title Page
List of Contributors
Preface
List of Abbreviations
1 Biofuels: The Back Story
1. Summary
2. 1.1 Introduction
3. 1.2 Some History
4. 1.3 Fossil Fuels
5. 1.4 Fossil Fuels and Carbon Dioxide
6. 1.5 Alternative Energy Sources
7. 1.6 Biofuels
8. Selected References and Suggestions for Further Reading
2 Biofuels in Operation
1. Summary
2. 2.1 Fuels for Transport
3. 2.2 Future Trends in Fuels Requirements and Technology
4. 2.3 Engines and Fuels – Progress vs Inertia
5. 2.4 Engine Constraints, Fuel Specifications and Enhanced Performance
6. 2.5 Biofuels – Implications and Opportunities
7. 2.6 Advanced Biofuels as Alternativesto Ethanol and FAME
8. 2.7 Biofuels for Aviation; ‘Biojet’
9. 2.8 Impact of Future Trends in Engine Design on Retail Biofuels
10. 2.9 Conclusion
11. Selected References and Suggestions for Further Reading
3 Anaerobic Digestion
1. Summary
2. 3.1 History and Development of Anaerobic Digestion
3. 3.2 Anaerobic Digestion: The Process
4. 3.3 Commercial Applications and Benefits
5. 3.4 Ethanol Production Linked with Anaerobic Digestion
6. 3.5 Financial and Economic Aspects
7. 3.6 UK and US Government Policies and Anaerobic Digestion – An Overview
8. 3.7 Concluding Comments
9. Selected References and Suggestions for Further Reading
4 Plant Cell Wall Polymers
1. Summary
2. 4.1 Nature and Biological Roles of Primary and Secondary Cell Walls
3. 4.2 Polysaccharide Composition of Primary and Secondary Cell Walls
4. 4.3 Post‐synthetic Modification of Cell‐wall Polysaccharides
5. 4.4 Polysaccharide Biosynthesis
6. 4.5 Non‐polysaccharide Components of the Plant Cell Wall
7. 4.6 Conclusions
8. Acknowledgements
9. Appendix
10. Selected References and Suggestions for Further Reading
5 Ethanol Production from Renewable Lignocellulosic Biomass
1. Summary
2. 5.1 Brief History of Fuel‐Ethanol Production
3. 5.2 Ethanol Production from Sugar Cane and Corn
4. 5.3 Lignocellulosic Biomass as Feedstocks for Ethanol Production
5. 5.4 Summary
6. 5.5 Examples of Commercial Scale Cellulosic Ethanol Plants
7. Selected References, Suggestions for Further Reading and Useful Websites
6 Fatty Acids, Triacylglycerols and Biodiesel
1. Summary
2. 6.1 Introduction
3. 6.2 Synthesis of Triacylglycerol
4. 6.3 Productivity
5. 6.4 Sustainability
6. 6.5 More Recently Exploited and Novel Sources of Lipids for Biofuels
7. 6.6 Concluding Remarks
8. Selected References and Suggestions for Further Reading
7 Development of Miscanthus as a Bioenergy Crop
1. Summary
2. 7.1 Introduction
3. 7.2 Developing Commercial Interest
4. 7.3 Greenhouse Gas Mitigation Potential
5. 7.4 Perspectives for ‘now’ and for the Future
6. Selected References and Suggestions for Further Reading
8 Mangrove Palm, Nypa fruticans: ‘3‐in‐1’ Tree for Integrated Food/Fuel and Eco‐Services
1. Summary
2. 8.1 Introduction: The ‘Food vs Fuel’ and ‘ILUC’ Debates
3. 8.2 Integrated Food‐Energy Systems (IFES): A Potential Solution
4. 8.3 Land Use: The Importance of Forest Ecosystem Services
5. 8.4 Sugar Palms: Highly Productive Multi‐Purpose Trees
6. 8.5 Nipa (Nypa fruticans): A Mangrove Sugar Palm with Great Promise
7. 8.6 Conclusion
8. Selected References and Suggestions for Further Reading
9 The Use of Cyanobacteria for Biofuel Production
1. Summary
2. 9.1 Essential Aspects of Cyanobacterial Biology
3. 9.2 Commercial Products Currently Derived from Cyanobacteria
4. 9.3 Cyanobacteria Culture
5. 9.4 Cyanobacterial Genomes and Genetic Modification for Biofuel Production
6. 9.5 Industrial Production of Biofuels from Cyanobacteria
7. 9.6 Conclusion
8. Selected References and Suggestions for Further Reading
10 Third‐Generation Biofuels from the Microalga, Botryococcus braunii
1. Summary
2. 10.1 Botryococcus braunii
3. 10.2 Microbial Interactions
4. 10.3 Botryococcus braunii as a Production Platform for Biofuels or Chemicals
5. 10.4 Improving Botryococcus
6. 10.5 Future Prospects and Conclusion
7. Selected References and Suggestions of Further Reading
11 Strain Selection Strategies for Improvement of Algal Biofuel Feedstocks
1. Summary
2. 11.1 Introduction
3. 11.2 Lipids in Microalgae
4. 11.3 Starch in Microalgae
5. 11.4 Metabolic Interconnection Between Lipid and Starch Biosynthesis
6. 11.5 Strategies for the Selection of Microalgae Strains with Enhanced Biofuel Feedstock Traits
7. 11.6 Conclusions
8. Acknowledgements
9. Selected References and Suggestions for Further Reading
12 Algal Cultivation Technologies
1. Summary
2. 12.1 Introduction
3. 12.2 Lighting
4. 12.3 Mixing
5. 12.4 Control Systems and Construction Materials
6. 12.5 Algal Production Systems at Laboratory Scale
7. 12.6 Algal Production in Open Systems
8. 12.7 Algal Production in Closed Systems
9. 12.8 Concluding Comments
10. Selected References and Suggestions for Further Reading
13 Biofuels from Macroalgal Biomass
1. Summary
2. 13.1 Macroalgal resources in the UK
3. 13.2 Suitability of macroalgae for biofuel production
4. 13.3 Biofuels from Macroalgae
5. 13.4 Future prospects
6. 13.5 Conclusion
7. Acknowledgements
8. Selected References and Suggestions for Further Reading
14 Lipid‐based Biofuels from Oleaginous Microbes
1. Summary
2. 14.1 Introduction
3. 14.2 Microalgae
4. 14.3 Oleaginous Yeasts
5. 14.4 Feedstocks for Heterotrophic Microbial Cultivation
6. 14.5 The Biochemical Process of Lipid Accumulation in Oleaginous Yeast
7. 14.6 Lipid Profile of Oleaginous Microbes
8. 14.7 Lipid Extraction and Processing
9. 14.8 Concluding Comments
10. Selected References and Suggestions for Further Reading
15 Engineering Microbial Metabolism for Biofuel Production
1. Summary
2. 15.1 Introduction
3. 15.2 Designer Biofuels
4. 15.3 Towards Industrialisation
5. 15.4 Conclusion
6. Selected References and Suggestions for Further Reading
16 The Sustainability of Biofuels
1. Summary
2. 16.1 Introduction
3. 16.2 Bioenergy policies
4. 16.3 Economics of bioenergy markets
5. 16.4 Environmental issues
6. 16.5 Life Cycle Assessment
7. 16.6 Conclusions
8. Selected references and suggestions for further reading
17 Biofuels and Bioenergy – Ethical Aspects
1. Summary
2. 17.1 Introduction to Ethics
3. 17.2 Biofuels and Bioenergy – Ethical Background
4. 17.3 The Key Ethical Issues
5. 17.4 Concluding comment
6. Selected references and suggestions for further reading
18 Postscript
1. Selected References and Suggestions for Further Reading
Index
End User License Agreement

List of Tables

Chapter 02
1. Table 2.1 Ethanol specifications in EU and US. Note these are subject to change and many other countries have their own specifications. Within the US, California has its own denatured ethanol specifications, 120% of which differ slightly from those in this chart. The EU specifications cover all 28 member states and also apply to European‐based EFTA countries (Iceland, Liechtenstein, Norway and Switzerland).
2. Table 2.2 Biodiesel specifications in EU and US. Note these are subject to change and many other countries have their own specifications. The EU specifications cover all 28 member states and also apply to European‐based EFTA countries (Iceland, Liechtenstein, Norway and Switzerland).
Chapter 04
1. Table 4.1 Major polysaccharides of ‘the’ land‐plant cell wall. For further details, see Fry (2011).
2. Table 4.2 Repeat units of xyloglucan. Code letters used for reporting xyloglucan sequences. Oligosaccharides are named by listing the code letters in sequence along the (1→4)‐β‐D‐glucan backbone of xyloglucan, from non‐reducing to reducing end. Square brackets indicate branching. From Franková and Fry (2012).
3. Table 4.3 Major cell wall‐localised glycanase activities found in plants.
4. Table 4.4 Major cell wall‐localised glycosidase activities found in plants.
Chapter 05
1. Table 5.1 Actual vs calculated theoretical ethanol yield using traditional Saccharomyces cerevisiae as the fermenting organism. Component content presented on a % weight basis.
2. Table 5.2 Theoretical yield estimates for selected feedstocks.
Chapter 06
1. Table 6.1 Lipid content as percentage of dry weight of a range of lipid‐rich seeds.
2. Table 6.2 Percentage fatty acid composition of triacylglycerols in storage lipids.
3. Table 6.3 Productivity of selected oil crops, litres/hectare.
4. Table 6.4 Productivity of selected ethanol crops, litres/hectare.
5. Table 6.5 ‘Energy content’ of fuels, KJ/kg.
Chapter 11
1. Table 11.1 Summary of studies for microalgae biofuel improvement via genetic mutagenesis.
Chapter 13
1. Table 13.1 Different sources of macroalgae for bioenergy generation.
Chapter 14
1. Table 14.1 Lipid content and fatty acid profile for selected oleaginous yeast and algal species.
Chapter 16
1. Table 16.1 The highest risks (C) where most environmental damage to occur comes from traditional agriculture. Lower risks (A) are achieved through innovative systems or perennial cropping.

List of Illustrations

Chapter 01
1. Figure 1.1 Haze over Singapore 2013.
2. Figure 1.2 (a) Hydro‐electric generating station at Lee Abbey Conference Centre, Devon. Even a small station like this can generate a significant proportion of the electricity used by up to 120 delegates plus 80 resident members of the community. This picture shows the turbine house and tail race through which the water is exiting. (b) Self‐cleaning Coanda screen at the head of the high pressure inlet pipe in the Lee Abbey system. (c) The generator in the turbine house at the Lee Abbey conference centre.
3. Figure 1.3 Wind Farm at Bears’ Down, Cornwall, UK.
4. Figure 1.4 Array of PV solar collectors installed by the Scottish dairy and food company, Mackie’s, at their farm in Aberdeenshire. With 7,000 panels, it is the largest array in Scotland, covering 4 hectares and generating 1.8 MW of electricity. When running at full capacity, it will reduce CO₂ emissions by about 850 tonnes/year.
Chapter 02
1. Figure 2.1 Fuel requirements are determined by numerous factors, categorised here under five broad headings, each evolving with time and impacting the nature of fuels required now and in the future.
2. Figure 2.2 The future biofuels landscape will evolve over time in response to technological advances in feedstocks, conversion processes, fuel/engine operations and market demand. Opportunities will arise from innovative developments, whilst critical constraints within the system generate inertia.
3. Figure 2.3 Fossil fuels may dominate until at least 2050. The proportion of conventional diesel is likely to increase relative to gasoline. The relative proportion of alternatives post‐2030 is subject to many evolving technological and other external factors so is less predictable.
4. Figure 2.4 Illustrative chain length and saturation profiles of various biodiesel types.
5. Figure 2.5 A sub‐set of plant oils yields chain lengths dominated by 10 to 14 carbons.
Chapter 03
1. Figure 3.1 Complete Mix Anaerobic Digester.
2. Figure 3.2 Pilot‐scale plug flow anaerobic digester, showing the easy loading ramp and the submerged digester.
3. Figure 3.3 Anaerobic digester integration with ethanol production.
4. Figure 3.4 The headline ’30 Mt less materials going into the economy’ is close to WRAP’s 2010 research: Securing the Future: The Role of Resource Efficiency 3.
Chapter 04
1. Figure 4.1 Fractionation of the cell wall into three major polysaccharide classes, and their physical properties.
2. Figure 4.2 Principal biological roles and monosaccharide building blocks of three major classes of cell‐wall polysaccharides.
3. Figure 4.3 Cartoons of representative segments of the major wall polysaccharides. The Greek letters (α or β) indicate the anomeric configuration of the sugar residue; the number represents the position on the adjacent sugar residue to which a glycosidic bond has been made. Fer = ferulic acid residue; – in circle = negative charge; arrow = apiose residue capable of forming a borate bridge; ⋅⋅⋅⋅⋅⋅ = continuation of polysaccharide backbone. From Fry et al. (2011).
4. Figure 4.4 Enzymic dissection of xyloglucan to release repeat units that can be readily purified and characterised. XEG = xyloglucan endo‐glucanase.
5. Figure 4.5 Overview phylogenetic tree of the streptophytes (land plants + charophytes). The restricted taxonomic distribution of selected wall components is highlighted: MLG only in the Poales and Equisetum; 3‐O‐methylrhamnose in the charophytes, bryophytes and homosporous lycophytes; 3‐O‐methylgalactose in the homosporous and heterosporous lycophytes.
6. Figure 4.6 Covalent vs non‐covalent cross‐links in the plant cell wall. Three types of non‐covalent cross‐link are: (a) hydrogen bonds, e.g. between a hemicellulose (e.g. xyloglucan) and a cellulosic microfibril; (b) ionic bonds between an anionic polymer (e.g. pectin) and a cationic polymer (e.g. extensin); (c) calcium‐bridges between two non‐esterified homogalacturonan domains. Non‐covalent bonds are individually weak but constitute strong cross‐links owing to their large number. Covalent cross‐links (d) are strong but few.
7. Figure 4.7 Four of the possible diferulates (dehydrodiferulates) found esterified to plant cell‐wall polysaccharides. R represents a long polysaccharide chain.
8. Figure 4.8 Oxidative coupling of tyrosine residues, as demonstrated in the cell‐wall glycoprotein, extensin. Reaction 1 may produce an inter‐ or intra‐polypeptide bridge; steric considerations show that Reaction 2 can only produce an inter‐polypeptide cross‐link.
9. Figure 4.9 The proposed formation of amide (isopeptide) cross‐links between pectic homogalacturonan domains via a bifunctional amine such as putrescine. Theoretically this reaction could be catalysed by a PME acting in transacylation mode. The by‐product, when PME catalyses either hydrolysis or transacylation, is methanol.
10. Figure 4.10 Intermediary metabolism leading to the ‘activated’ sugars, nucleoside diphosphate sugars, the precursors of cell‐wall polysaccharides. Note that UDP‐GlcA and GDP‐Man could each potentially arise via two competing routes. Grey boxes (at left) indicate scavenger pathways, capable of bringing free monosaccharides (e.g. released during polysaccharide turnover, or experimentally added in radioactive form) back into central metabolism and thus de‐novo polysaccharide synthesis. Comparable scavenger pathways do not exist for xylose, rhamnose or apiose. Numbered enzymes in the primary pathways (bold arrows) are: 1. sucrose synthase; 2. invertase; 3. hexokinase; 4. fructokinase; 5. UDP‐Glc 4‐epimerase; 6. UDP‐Glc pyrophosphorylase; 7. phosphoglucomutase; 8. phosphoglucose isomerase; 9. UDP‐Glc dehydrogenase; 10. UDP‐Rha synthase; 11. UDP‐GlcA 4‐epimerase; 12. UDP‐GlcA decarboxylase; 13. UDP‐Api synthase; 14. GDP‐Glc pyrophosphorylase; 15. GDP‐Glc 2‐epimerase; 16. GDP‐L‐Gal synthase (GDP‐Man 3,5‐epimerase); 17. GDP‐Fuc synthase (two enzymes); 18. myo‐inositol phosphate synthase; 19. myo‐inositol phosphatase; 20. myo‐inositol oxygenase; 21. glucuronokinase; 22. UDP‐GlcA pyrophosphorylase; 23. phosphomannose isomerase; 24. phosphomannomutase; 25. GDP‐Man pyrophosphorylase; 26. UDP‐Ara mutase. Enzymes in the scavenger pathways are ATP‐dependent kinases (k) and UTP‐ or GTP‐dependent pyrophosphorylases (p).
11. Figure 4.11 An experimental strategy for quantitatively estimating which of two competing pathways actually contribute(s) to wall polysaccharide biosynthesis in cases where competing pathways potentially exist (cf. Figure 4.10). Living plant cells, grown with glycerol as major carbon source, are simultaneously fed [³H]galactose (white rectangle) and [¹⁴C]fructose (black rectangle). As the two isotopes mingle in the intermediates, different ³H:¹⁴C ratios are established (represented by various shades of grey between the ‘white’ and ‘black’ tracers), which are preserved in the polysaccharide end‐products. The isotope ratio in the end‐product indicates from which intermediate it principally arose.
Chapter 05
1. Figure 5.1 (a) Henry Ford’s Model T. (b) Environmental awareness of industrial processes. (c) Closed pumps along Interstate 5 in Oregon in the United States, October 1973.
2. Figure 5.2 Global ethanol production in 2013 worldwide in millions of gallons by country, millions of gallons, and percentage of global production (data source USDA ‐FAS). Note: 1US gallon = 3.785 litres.
3. Figure 5.3 World ethanol production, millions of US gallons, from 2005–2008 with estimates up to 2017.
4. Figure 5.4 Dry mill ethanol process.
5. Figure 5.5 S. cerevisiae ethanol production with glucose as the carbon source or substrate.
6. Figure 5.6 Overall schematic for ethanol production from lignocellulosic biomass.
7. Figure 5.7 Disruption of pectin, hemicellulose, cellulose and lignin using physical and chemical pretreatment of biomass.
8. Figure 5.8 The production of inhibitory compounds from the various polymers of biomass during pretreatment.
9. Figure 5.9 Effects of certain inhibitory compounds on Saccharomyces cerevisiae. ‘Blocks’ indicate the inhibition of processes or enzymes, while arrows indicate damage or inhibition of cellular components without a specific known target.
Chapter 06
1. Figure 6.1 Synthesis of FAs in plants. Carbons are added two at a time, each pair being donated form malonyl‐CoA in the form of acetyl‐CoA.
2. Figure 6.2 The Kennedy pathway for synthesis of triacyl glycerol.
3. Figure 6.3 Formation of oleosomes.
Chapter 07
1. Figure 7.1 Geographical distribution of the major Miscanthus species. The distribution of M. x giganteus is not fully known, but can potentially be found in regions where M. sinensis and M. sacchariflorus overlap.
2. Figure 7.2 Flowers of Miscanthus sinensis.
3. Figure 7.3 Miscanthus growing at Aberystwyth, UK with Dr Elaine Jensen and Dr John Clifton‐Brown.
4. Figure 7.4 Dried and compressed Miscanthus on sale for domestic heating at a farm shop near Exeter, UK
5. Figure 7.5 Distribution of Switchgrass, Panicum virgatum, in the USA and Canada.
Chapter 08
1. Figure 8.1 Sustainable food and fuel production.
2. Figure 8.2 Ethanol production (litres/ha/yr).
Chapter 09
1. Figure 9.1 Above: transmission electron micrograph of a spherical cyanobacterium, with annotated structures. Below: Diagrammatic representation of the key metabolic pathways for the production of carbohydrates and biofuel molecules in cyanobacteria.
2. Figure 9.2 Diagrammatic representation of the process of generating unmarked knockout mutations for cyanobacteria.
Chapter 10
1. Figure 10.1 Brightfield Micrograph of Botryococcus braunii. B. braunii is a colonial microalga. Colonies are formed of adjoined, pyriform algal cells (A). The green colour is from the single, lobed chloroplast (C) that is present is each cell. Cells also contain a number of cytoplasmic vesicles (V) that contain the characteristic hydrocarbons synthesised by the algae. The hydrocarbons are secreted into the extracellular matrix (ECM) and, when placed under a microscope slide, ooze out of the colony in droplets (HC). The scale bar represents 10 µm.
2. Figure 10.2 Botryococcus braunii hydrocarbons. Structures of the principle hydrocarbons synthesised by the different ‘chemical races’ of B. braunii.
3. Figure 10.3 Association between Botryococcus braunii and bacteria. The left panel is a scanning electron micrograph of a single B. braunii colony. The algal cells are barely visible and appear shrouded by the layer of secreted hydrocarbons. The right panel shows a scanning electron micrograph of a B. braunii colony from which the hydrocarbons have been removed. The algae appear as flattish pear‐shaped cells; note a number of rod‐shaped bacterial cells that are closely associated with the surface of the algae. The scale‐bars represent 5 µm.
4. Figure 10.4 Potential synthetic biology of Botryococcus braunii hydrocarbon synthesis. The B. braunii isoprenoid biosynthetic pathway of B. braunii may be leveraged to produce advanced biofuels through the application of synthetic biology approaches. Enzyme functions are indicated in boxes, and conversions are indicated by arrows. SSL, squalene synthase like enzymes 1, 2 and 3; S‐AM, S‐adenosyl methonine transferase; FS, farnesene synthase; BIS, bisbolene synthase; PS, pinene synthase; LS, limonene synthase; ispS, isoprene synthase.
Chapter 11
1. Figure 11.1 Reiterative and transgressive selection by fluorescence‐activated cell sorting (FACS). Fluorescently stained microalgae cell populations are collected based upon their fluorescence intensity using FACS. After collection, cells with either greater or lesser fluorescence intensity are collected, re‐cultured in an aerated flask, and iteratively selected on a weekly basis until significant differences in fluorescence are evident. Cultures are analysed at regular intervals for starch and lipid content.
2. Figure 11.2 Reiterative and transgressive selection by continuous buoyant density gradient centrifugation (BDGC). Microalgae cells are placed atop a continuous Percoll® density gradient and centrifuged until cells reach their buoyant density equilibrium. After centrifugation, cell populations with either greater or lesser buoyant densities are removed, re‐cultured in an aerated flask, and iteratively selected on a weekly basis until significant differences in buoyant density are evident. Cultures are analyzed at regular intervals for starch and lipid content.
Chapter 12
1. Figure 12.1 Examples of common lighting arrangements. (a) fluorescent light tubes; (b) a Metal Halide lamp; and (c) an LED lighting block with red/blue lighting.
2. Figure 12.2 Photos of different mixing systems, (a) Turbines are often deployed to create radial mixing within stirred tank reactors. (b) Paddlewheel, used to create directional fluid mixing. (c) Bubble column reactors, where turbulence is created by direct bubbling of the culture. (d) ALR, where air injection creates directional liquid circulation. (e) Velocity pump, clearly showing the inlet and outlet.
3. Figure 12.3 AlgaeConnect control system and pH probe. The probes can interface with control systems such as AlgaeConnect produced by AlgaeLab Systems.
4. Figure 12.4 Example of a flask culture, and shaker arrangement. Cotton wool is used to allow for gaseous exchange whilst preventing the entrance of contaminants. The flasks are placed on rotational shakers to provide adequate mixing for growth.
5. Figure 12.5 Example of a flask culture, and shaker arrangement. Diagram illustrates the components of a stirred fermenter, note submerged aerator and impeller. Lights can be arranged around the reactor or submerged inside, as long as they do not interfere with the circulation of the impeller.
6. Figure 12.6 Schematic and photograph showing the typical arrangement of a raceway pond. (a) Shows an aerial view, indicating how the pond is mixed and sparged with CO₂; and (b) Typical raceway pond.
7. Figure 12.7 Schematic diagram and photograph of a membrane PBR. The photograph on the right shows the strains Nannochloropsis (nc), Isochrysis (is) and Tetraselmis (ts) grown within drip fed membrane reactors.
8. Figure 12.8 Diagram showing the potential arrangements of flat panelled reactors. (a) Gives an indication of the many different tilt angles that are possible to optimise solar penetration. (b) Shows an array of flat panel bioreactors.
9. Figure 12.9 Schematic showing the potential arrangements of tubular reactors. (a) shows a manifold system, illustrating how the manifolds split the main flow. Modified from Chisti (2007). (b) Varicon Aqua’s BioFence™, which operates under a manifold system (c) shows a serpentine arrangement (Posten, 2009); panel (d) shows such an arrangement in use in a Varicon Aqua Phyco‐Flow in Hawaii.
10. Figure 12.10 Bubble columns. (a) The diagram indicates the relative sections of light and dark zonation within the bubble column. It also shows how air bubbles can deform as they rise through the column. (b) A battery of bubble columns.
11. Figure 12.11 Different configurations of airlift bioreactors. (a) Split‐cylinder internal‐loop; (b) concentric draught‐tube internal‐loop; (c) draught‐tube internal‐loop with vertically split draught‐tube; and (d) external loop.
12. Figure 12.12 Diagram shows the potential arrangements of ALRs. (a) and (b) show how an airlift system can be created by directing the flow of the airlift. Images reproduced, with permission, from Vunjak‐Novakovic et al. (2005). (c) shows the ALR designed and built by Varicon Aqua Solutions in collaboration with University College London.
Chapter 13
1. Figure 13.1 Main carbohydrates in a typical kelp, Laminaria hyperborean.
Chapter 14
1. Figure 14.1 Triglyceride biosynthesis pathway from glucose to triglycerides in oleaginous yeast. AAT: ATP‐ADP translocase; ACL: ATP‐citrate lyase; AS: ATP synthase; DIC: dicarboxylate carrier; MDH: malate dehydrogenase; ME1: malic enzyme; PC: pyruvate carboxylase; PDH: pyruvate dehydrogenase; PT: pyruvate transporter; TIC: tricarboxylate carrier
2. Figure 14.2 Schematic of intracellular triglyceride synthesis via the α‐glycerol phosphate acylation pathway. DAG: Diacylglycerol; DAGAT: Diacylglycerol acyltransferase; G‐3‐P: Glycerol‐3‐phosphate; GAT: G‐3‐P acyltransferase; lpa: lysophosphatidic acid; LPAAT: Lysophosphatidic acid acyltransferase; PA: Phosphatidic acid; PAP: Phosphatidic acid acyltransferase; TAG: Triacylglycerol. See also Chapters 6 and 14.
Chapter 15
1. Figure 15.1 Metabolic pathways to isoprenoid‐derived fuels. (a) Two alternative pathways generate the starter molecules IPP (isopenetyl diphosphate) and DMAPP (dimethylallyl diphosphate): via mevanolate (the MEV pathway; cytosolic) or via 2‐C‐methyl‐D‐erythritol‐4‐phosphate (the MEP pathway; plastidial). IPP and DMAPP are condensed to form geranyl pyrophosphate, which may then be converted to C₁₀ hydrocarbons, or may undergo further rounds of extension with IPP prior to conversion to C₁₅ and C₂₀ hydrocarbons, via the activities of terpene synthases (RS). (b) Condensation of IPP and DMAPP to form geranyl pyrophosphate and subsequent elongation with another molecule of IPP to form farnesyn pyrophosphate. At each step, a pyrophosphate molecule is released.
2. Figure 15.2 Metabolic pathways to higher alcohols. (a) Fermentative routes to higher alcohol (isopropanol) production introduced into E. coli. Thl (thiolase), CtfAB (coenzyme A transferase, A and B subunits) and ADC (acetoacetate decarboxylase) from C. acetobutylicum, ADH (butanol dehydrogenase) from Clostridium beijerinckii and over‐expression of E. coli AtoAD (acetoacetyl‐CoA transferase). C. beijerinckii 1‐butanol biosynthesis pathway. Thl (thiolase), Hbd (3‐hydroxybutyrl‐CoA dehydrogenase), Crt (cronotase), Bcd (butyrl‐CoA dehydrogenase), AdhE2 (butaraldehyde/butanol dehydrogenase). In the synthetic butanol pathway PhaA (thiolase from Ralstonia eutropha) replaced Thl, PhaB (acetoacetyl‐CoA reductase from R. eutropha) replaced Hbd, Ccr (Cronotyl‐CoA reductase (encoded by Ter) from Treponema denticola) replaced Bcd and EefA & EtfB. (b) Non‐fermentative routes in higher alcohols. 2‐ketoacid‐derived short chain alcohol biosynthesis utilises substrates from amino acid biosynthesis (2‐ketoacids) derived from a common pyruvate pool. Ketoacid decarboxylases (KDC) and alcohol dehydrogenases (ADH) catalyse the 2‐step conversion form ketoacid to aldehyde, and from aldehyde to corresponding alcohol.
3. Figure 15.3 Metabolic pathways of FA metabolism. FA biosynthesis commences through the condensation of acetyl‐CoA with malonyl‐ACP. This creates a ketoacyl‐ACP molecule that can be cyclic‐elongated through the sequential addition of further malonyl‐moieties at 2‐carbon increments. Free FAs may be liberated through the action of thioesterases (Te) with different chain length specificities, a modification which also serves to increase the FA pool size. FA titres have also been increased by over‐expression of ACC (acetyl‐CoA carboxylase) and removal of flux into the ß‐oxidation pathway using either fadD (ACS; acyl‐Co‐A synthetase) or fadE (acyl‐CoA dehydrogenase) knockouts. Conversely, over‐expression of ACS with ΔfadE serves to increase fatty acyl‐CoA pools for FAEE production. Expression of branched chain ketoacid dehydrogense (BCKD) and KASIII from Bacillus subtilis can be used for the production of branches chain FAs in E. coli as branched CoA primer units are incorporated in FA biosynthesis.
4. Figure 15.4 Metabolic pathways to petroleum‐replica biofuels. Natural and synthetic alkane biosynthesis pathways have been recreated in E. coli and in yeast. The initial elucidation of a two‐step alkane route to alkanes, via (AAR) acyl‐ACP reductase and ADO (aldehyde deformylase) was demonstrated by expressing these genes in E. coli. Likewise, bacterial production of alpha‐olefins via Ole T from Jeotgalicoccus spp. Exploitation of FAs, rather than fatty acyl‐ACP using FAR (FA reductase complex from Photorahbus luminescens), allowed alkane output to be determined by the predominant FA species. A similar approach of producing alkanes directly from FAs was reported for CAR (carboxylic acid reductase from Mycobacterium marinum) though this report was principally aimed at tailoring fatty alcohol content. A three gene cluster (Mlut_ 13230‐133250) from Micrococcus luteus, produces VLC alkenes when expressed in E. coli. The cer1/cer3 pathway has been genetically identified from Arabidopsis thaliana and expressed in yeast to produce VLC alkanes (>C₃₀). Alkanes and alkenes of this chain length are not useful as fuel molecules per se, but knowledge of these pathways may help with engineering tailored synthetic alkanes routes.
5. Figure 15.5 Strategies for improving the chances of industrialisation. Alternative sources for energy and carbon skeletons for biofuel synthesis include photosynthetic carbon fixation by algae and cynaobacteria; secretion of digestive enzymes for the digestion of waste biomass (typically E. coli) or electrochemical formate production (Ralstonia eutropha). Within the cell enhanced or synthetic metabolic pathways for designer biofuels may be co‐localised using scaffolding techniques (E. coli) or through sub‐cellular compartmentalisation of different organelles (S. cerevisiae) and may be placed under dynamic regulatory responses such as the FadR transcription factor (E. coli) that respond to the activity of the biofuel pathway. Recovery of the fuel molecule, removal of toxic compounds and alleviation of feedback inhibition may be aided by the use of efflux pumps to export the end product from the cell (E. coli).
6. Figure 15.6 Novel sources of energy and carbon skeletons. Researchers at UCLA have reported two alternative strategies for generating the energy and carbon skeletons required for biofuel molecules. (a) E. coli was engineered to produce higher alcohol biofuels from protein hydrosylates. 1) E. coli was engineered by chemical mutagenesis to accept a wider range of amino acids as the sole carbon source rather than wild type E. coli. 2) Three transamination and deamination cycles are introduced as well as 3) the removal of GdhA and GlnA to prevent re‐assimulation of ammonia. 4) Pyruvate is converted to the various 2‐ketoacids by endogenous LeuABCD and llvHCD activity, or through over‐expression of AlsS and llvCD. 5). Final conversion to higher alcohols follows the reactions shown in Figure 15.2 catalysed by introduced ketoacid decarboxylase (KDC) and alcohol dehydrogenase (ADH). The authors report 56% theoretical maximum yield and the ability to utilise a range of different sources, including microalgae, yeast and bacterial feedstock. (b) Chemolithotrophic bacteria fix CO₂ using NADH, indepentent of the light reactions found in plant and cyanobacteria. One such bacteria, R, eutropha, was engineering to produce isobutanol, while endogenous pathways to polyhydroxybutyrate were removed. Genetically‐modified R. eutropha were also used to identify O_2‐ and no stress as a limiting factor on cell growth, which was attributed to anode chemistry upon application of electrical currents to the media. Consequently, an inexpensive ceramic shield was used to quench O_2‐ and NO diffusion from the anode, and cell growth was no longer inhibited. Isobutanol was produced at 140 mg/L from electricity and CO₂.
Chapter 16
1. Figure 16.1 Land requirements for biofuels.
2. Figure 16.2 Factors which need to be considered in realising the potential of bioenergy.
Chapter 17
1. Figure 17.1 Aerial photo of the lands taken by Addax Bioenergy for its sugar cane plantation in Sierra Leone.

Preface

A century ago, petroleum – what we call oil – was just an obscure commodity; today it is almost as vital to human existence as water.

James Buchan, Political commentator and author

The use of vegetable oils for engine fuels may seem insignificant today. But such oils may become in course of time as important as petroleum and the coal tar products of the present time.

Rudolf Diesel, engineer and inventor of the compression engine (quotation dates from 1912)

Most people have difficulty coming to grips with the sheer enormity of energy consumption.

Rex Tillerson, Civil engineer, businessman and President/CEO of the Exxon‐Mobil Corporation

We have to rethink our whole energy approach, which is hard to do because we're so dependent on oil, not just for fuel but also plastic … We have to think quite carefully about using oil and its derivatives, because it's not going to be around forever.

Margaret Atwood, Author, literary critic and environmental activist

We can no longer allow America’s dependence on foreign oil to compromise our energy security. Instead, we must invest in inventing new ways to power our cars and our economy. I’ll put my faith in American science and ingenuity any day before I depend on Saudi Arabia.

Senator John Kerry, US Secretary of State, 2013–2016

There is an urgent need to stop subsidizing the fossil fuel industry, dramatically reduce wasted energy, and significantly shift our power supplies from oil, coal, and natural gas to wind, solar, geothermal, and other renewable energy sources.

Bill McKibben, Author, educator and environmental activist

These quotations provide a nice series of snapshots. The world is energy‐hungry and increasingly so. The need for fuels for transport makes up a large proportion of that hunger. That need is largely met by petroleum (literally ‘rock‐oil’), 70% of which is used for transport by road, air or sea, but there are issues related to its continued use and availability (even if concerns about ‘peak oil’ – the moment when global oil production reaches its maximum – have declined somewhat). There are concerns that some developed countries’ need for oil may make them economic or moral hostages to countries that are oil producers. And above all is the realisation that burning fossil fuels (principally coal, natural gas and oil) is the major contributor to anthropogenic climate change. There is thus a drive to develop renewable sources of energy, sources of energy that do not involve burning fossil fuels. And we have to say, as is evident from Chapter 1, there has been very good progress in generation of electricity via environmental energy sources. Electricity can of course be used to power some forms of transport. but it still glaringly obvious that transport is very dependent on oil (and to a lesser extent, coal and gas), and will continue to be dependent on liquid fuels well into the future. So we come to this book, which deals with current areas of research aimed at finding ways of using renewable biological resources to provide fuels mainly for transport, research which becomes ever more urgent as the reality of climate change becomes more apparent.

Biofuels, therefore, should be viewed in the context of sustainability, either as alternatives to reduce petroleum use during the transition to other forms of transport, energy or primary materials, or as a way to mitigate climate change. The use of petroleum distillate in mass transport did not happen overnight (indeed petrol was once considered a waste product of oil refining); likewise, biofuels are at the very early stages of development. Biofuels research is intense, with new options being imagined and solutions being proposed almost weekly. Every new technology explores a previously unimagined design landscape. The issue with biofuels is that technical developments are heavily constrained by existing infrastructure, land use and global commerce in commodities. As yet, we cannot pick the future biofuel ‘winners’, but any biofuel solution (and there may be several) must be responsive to a number of criteria, including cost, technical feasibility, efficiency, reliability, sustainability and, arguably most difficult of all, our lifestyle expectations.

We are bound to say that the book has been a long time coming. It is actually several years since a conversation in an Oxford coffee shop between JAB and Rachel Wade of Wiley‐Blackwell led to the idea of an edited text on Biofuels. It took a long time to recruit authors and even then, other factors outside the control of editors and publisher, led to further delays and loss of some of the planned and contracted chapters. Against this background, we are especially grateful to those authors who have remained with the project and provided the excellent and interesting range of chapters presented here. A number of them have been remarkably patient as they waited for news of further progress after submitting their chapters. We are also grateful to colleagues who have given us their time in discussion and/or provided diagrams and figures for us to use. JAB expresses special thanks to Dr David Stafford of Enviro‐Control Ltd, Professor Jim Lynch of the University of Surrey and Professor Steve Hughes of the University of Exeter for their long‐term friendship, support and readiness to share their knowledge and expertise; also to environmental engineer, Rachel Oates of the Lee Abbey Community, Devon, for her knowledgeable enthusiasm and readiness to talk about environmental energy sources, especially ‘micro‐hydro’ (see Chapter 1). JL is especially indebted to Professor Rob Lee of Royal Dutch Shell, his ‘partner in slime’ for the past 15 years and to Drs Mike Goosey and Jeremy Shears, both directors of Shell Biodomain, for their positive and supportive vision of open innovation between academia and industry to solve global problems. Heartfelt thanks also to all of the current and past members of the Exeter Microbial Biofuels Group for their talent, commitment, professionalism and humour, to our numerous collaborators in academia and in industry, and to the BBSRC for supporting our research. Our programme would not be possible without the direct support of the University of Exeter. JL is personally grateful to our Vice Chancellor Professor Sir Steve Smith and to our Deputy Vice‐Chancellor for research, Professor Nick Talbot, FRS (formerly his head of department) for their genuine and sustained interest, and to his all colleagues in professional services, notably Linda Peka and Caroline Hampson, for their immeasurable patience in underpinning our research.

Finally, we want to say a big ‘Thank You’ for the patience of our publishers at Wiley‐Blackwell and especially those closely involved with this book, Rachel Wade at Oxford who helped to initiate the project, Fiona Seymour at Chichester who was for a long time ‘our’ editor, Audrie Tan at Singapore who took over from Fiona as the last few chapters came in and Vinodhini Mathiyalagan together with Shummy Metilda who supervised the book’s production. It would have been so easy for these individuals and for Wiley‐Blackwell themselves to abandon the book as we sought yet another re‐scheduling. We are very grateful that they stuck with us.

John Love
John A. Bryant
Exeter, August 2015