Biomass to Biofuels

Biomass to Biofuels: Strategies for Global Industries
Edited by 

ALAIN A. VERTES Sloan Fellowship, London Business School, UK NASIB QURESHI United States Department of Agriculture, National Center for Agricultural Utilization Research, Peoria, Illinois, USA HANS P. BLASCHEK University of Illinois at Urbana-Champaign, USA HIDEAKI YUKAWA Research Institute of Innovative Technology for the Earth, Kyoto, Japan

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Foreword Preface Contributors Part I: Structure of the Bioenergy Business 1 Characteristics of Biofuels and Renewable Fuel Standards Alan C. Hansen, Dimitrios C. Kyritsis and Chia fon F. Lee
1.1 1.2 1.3 1.4 1.5 1.6 Introduction Molecular Structure Physical Properties Chemical Properties Biofuel Standards Perspective References

xi xiii xix 1 3
3 4 5 10 17 19 21


The Global Demand for Biofuels: Technologies, Markets and Policies J€rgen Scheffran u
2.1 2.2 2.3 2.4 2.5 2.6 Introduction Motivation and Potential of Renewable Fuels Renewable Fuels in the Transportation Sector Status and Potential of Major Biofuels Biofuel Policies and Markets in Selected Countries Perspective References

27 29 31 34 37 44 50


Biofuel Demand Realization Stephen R. Hughes and Nasib Qureshi
Introduction Availability of Renewable Resources to Realize Biofuel Demand Technology Improvements to Enhance Biofuel Production Economics US Regulatory Requirements for Organisms Engineered to Meet Biofuel Demand 3.5 Perspective Acknowledgments References 3.1 3.2 3.3 3.4

55 56 59 64 67 68 68


Advanced Biorefineries for the Production of Fuel Ethanol Stephen R. Hughes, William Gibbons and Scott Kohl
4.1 4.2 Introduction Ethanol Production Plants Using Sugar Feedstocks

71 73


Contents 4.3 Dedicated Dry-Grind and Dry-Mill Starch Ethanol Production Plants 4.4 Dedicated Wet-Mill Starch Ethanol Production Plants 4.5 Dedicated Cellulosic Ethanol Production Plants 4.6 Advanced Combined Biorefineries 4.7 Perspective Acknowledgments References 75 79 81 83 84 86 86

Part II: Diesel from Biomass 5 Biomass Liquefaction and Gasification Nicolaus Dahmen, Edmund Henrich, Andrea Kruse and Klaus Raffelt
5.1 5.2 5.3 5.4 Introduction Direct Liquefaction Biosynfuels from Biosyngas Perspective References

89 91
91 92 104 116 118


Diesel from Syngas Yong-Wang Li, Jian Xu and Yong Yang
6.1 6.2 6.3 6.4 6.5 6.6 Introduction Overview of Fischer–Tropsch Synthesis Historical Development of the Fischer–Tropsch Synthesis Process Modern Fischer–Tropsch Synthesis Processes Economics Perspective Acknowledgments References

123 124 125 128 135 136 137 138


Biodiesel from Vegetable Oils Jon Van Gerpen
7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8 Introduction Use of Vegetable Oils as Diesel Fuels Renewable Diesel Properties Biodiesel Production Transesterification Biodiesel Purification Perspective References

141 142 146 146 151 152 156 158 160


Biofuels from Microalgae and Seaweeds Michael Huesemann, G. Roesjadi, John Benemann and F. Blaine Metting
8.1 8.2 8.3 8.4 Introduction Biofuels from Microalgae: Products, Processes, and Limitations Biofuels from Seaweeds: Products, Processes, and Limitations Perspective References

165 167 174 179 180



Part III: Ethanol and Butanol 9 Improvements in Corn to Ethanol Production Technology Using Saccharomyces cerevisiae Vijay Singh, David B. Johnston, Kent D. Rausch and M.E. Tumbleson
9.1 9.2 9.3 9.4 9.5 9.6 9.7 9.8 Introduction Current Industrial Ethanol Production Technology Granular Starch Hydrolysis Corn Fractionation Simultaneous SSF and Distillation Dynamic Control of SSF Processes Cost of Ethanol Perspective References


187 187 191 192 193 194 195 196 196


Advanced Technologies for Biomass Hydrolysis and Saccharification Using Novel Enzymes Margret E. Berg Miller, Jennifer M. Brulc, Edward A. Bayer, Raphael Lamed, Harry J. Flint and Bryan A. White
10.1 10.2 10.3 10.4 10.5 10.6 Introduction The Substrate Glycosyl Hydrolases The Cellulosome Concept New Approaches for the Identification of Novel Glycoside Hydrolases Perspective References


199 200 200 203 205 209 209


Mass Balances and Analytical Methods for Biomass Pretreatment Experiments Bruce S. Dien
11.1 11.2 11.3 11.4 11.5 11.6 11.7 Introduction Analysis of Feedstocks for Composition and Potential Ethanol Yield Pretreatment Enzymatic Extraction of Sugars Fermentation of Pretreated Hydrolysates to Ethanol Feedstock and Process Integration Perspective Acknowledgments References

213 214 218 224 226 228 229 229 230


Biomass Conversion Inhibitors and In Situ Detoxification Z. Lewis Liu and Hans P. Blaschek
12.1 12.2 12.3 12.4 12.5 12.6 12.7 12.8 Introduction Inhibitory Compounds Derived from Biomass Pretreatment Inhibitory Effects Removal of Inhibitors Inhibitor-Tolerant Strain Development Inhibitor Conversion Pathways Molecular Mechanisms of In Situ Detoxification Perspective Acknowledgments References

233 234 239 242 243 245 247 253 254 254




Fuel Ethanol Production From Lignocellulosic Raw Materials Using Recombinant Yeasts Grant Stanley and B€rbel Hahn-H€gerdal a a
13.1 13.2 13.3 13.4 13.5 Introduction Consolidated Bioprocessing and Ethanol Production Pentose-Fermenting S. cerevisiae Strains Lignocellulose Fermentation and Ethanol Inhibition Perspective Acknowledgments References

261 265 271 275 281 282 282


Conversion of Biomass to Ethanol by Other Organisms Siqing Liu
14.1 14.2 14.3 14.4 14.5 Introduction Desired Biocatalysts for Biomass to Bioethanol Gram-Negative Bacteria Gram-Positive Bacteria Perspective Acknowledgments References

293 294 295 300 305 306 306


Advanced Fermentation Technologies Masayuki Inui, Alain A. Verts and Hideaki Yukawa e
15.1 15.2 15.3 15.4 15.5 15.6 15.7 15.8 15.9 Introduction Batch Processes Fed-Batch Processes Continuous Processes Immobilized Cell Systems Growth-Arrested Process Integrated Bioprocesses Consolidated Bioprocessing (CBP) Perspective References

311 312 314 315 317 319 322 324 325 327


Advanced Product Recovery Technologies Thaddeus C. Ezeji and Yebo Li
16.1 16.2 16.3 Introduction Membrane Separation Advanced Technologies for Biofuel Recovery: Industrially Relevant Processes 16.4 Perspective Acknowledgments References

331 333 336 343 343 343


Clostridia and Process Engineering for Energy Generation Nasib Qureshi and Hans P. Blaschek
17.1 17.2 17.3 17.4 17.5 Introduction Substrates, Cultures, and Traditional Technologies Agricultural Residues as Substrates for the Future Butanol-Producing Microbial Cultures Regulation of Butanol Production and Microbial Genetics

347 348 348 349 350

Contents 17.6 17.7 17.8 17.9 17.10 Novel Fermentation Technologies Novel Product Recovery Technologies Fermentation of Lignocellulosic Substrates in Integrated Systems Integrated or Consolidated Processes Perspective Acknowledgments References

ix 351 351 353 355 355 355 356

Part IV: Hydrogen, Methane and Methanol 18 Hydrogen Generation by Microbial Cultures Anja Hemschemeier, Katrin M€llner, Thilo R€hle and Thomas Happe u u
18.1 18.2 18.3 Introduction: Why Biological Hydrogen Production? Biological Hydrogen Production Metabolic Basics for Hydrogen Production: Fermentation and Photosynthesis 18.4 H2 Production in Application: Cases in Point 18.5 Perspective References

359 361
361 362 366 370 378 380


Engineering Photosynthesis for H2 Production from H2O: Cyanobacteria as Design Organisms Nadine Waschewski, Gbor Bernt, and Matthias R€gner a a o
19.1 19.2 19.3 The Basic Idea: Why Hydrogen from Water? Realization: Three Mutually Supporting Strategies The Biological Strategy: How to Design a Hydrogen-Producing (Cyano-) Bacterial Cell 19.4 Engineering the Environment of the Cells: Reactor Design 19.5 How Much Can We Expect? The Limit of Natural Systems 19.6 Perspective Acknowledgments References

387 388 390 395 397 397 399 399


Production and Utilization of Methane Biogas as Renewable Fuel Zhongtang Yu, Mark Morrison, and Floyd L. Schanbacher
20.1 20.2 20.3 20.4 20.5 20.6 20.7 Introduction The Microbes and Metabolisms Underpinning Biomethanation Potential Feedstocks Used for Methane Biogas Production Biomethanation Technologies for Production of Methane Biogas Utilization of Methane Biogas as a Fuel Perspective Concluding Remarks Disclaimer References

403 404 408 413 426 427 428 429 429


Methanol Production and Utilization Gregory A. Dolan
21.1 21.2 21.3 21.4 Introduction Biomass Gasification: Mature and Immature Feedstocks: Diverse and Plentiful Biomethanol: ICEs, FFVs, and FCVs

435 438 439 441

7 Introduction In-Fibril Modification In-Wall Modifications In-Planta Modifications In-CRES-T Modification A Catalogue of Gene Families for Glycan Synthases and Hydrolases Perspective Acknowledgments References 457 459 459 460 464 466 468 471 481 481 482 23 Axes of Development in Chemical and Process Engineering for Converting Biomass to Energy Alain A.3 23. Nobuyuki Nishikubo.6 21.8 Case Study: Waste Wood Biorefinery Case Study: Two-Step Thermochemical Conversion Process Case Study: Mobile Methanol Machine Case Study: Scandinavia Leading the Way with Black Liquor Methanol Production 21.6 22. Verts and Sarit Soccary Ben Yochanan e 24.3 Governmental Incentives to Support the Nascent Biofuel and Biomaterial Industry 24.9 Case Study: Methanol Fermentation through Anaerobic Digestion References 443 444 446 447 450 454 Part V: Perspectives 22 Enhancing Primary Raw Materials for Biofuels Takahisa Hayashi. Masaru Ohme-Takagi.1 23.5 22.2 23.1 24.4 22.7 21.5 Global Outlook Enhancement of Raw Material Biomass Conversion of Biomass to Fuels and Chemicals Chemical Engineering Development Perspective References 491 491 500 502 506 509 515 24 Financing Strategies for Industrial-Scale Biofuel Production and Technology Development Start-Ups Alain A.5 21.4 Perspective: What is the Best Funding Source for Each Step in a Company’s Development? References 523 523 530 538 540 543 Index 547 .4 23. Nobutaka Mitsuda.3 22. Verts e 23.2 22. Rumi Kaida.2 Background: The Financial Environment Biofuels Project: Steps in Value Creation and Required Funding at Each Stage 24. Shin-ichiro Kidou and Kouki Yoshida 22.1 22.x Contents 21.

the oil business became the petrochemicals industry that today touches every part of our lives. to a world where there is a greater diversity and balance with biofuels. it is important to learn the lessons from the petrochemical industry. Equally necessary are the issues of business drivers. the oil business went from a nascent industry that grew into an industrial complex completely ingrained into all aspects of the economy. Through the adoption of these design frameworks as a holistic system rather than individual criteria. Today. One can view these topics as the mortar that holds the building blocks of technology in place. The lessons provided in this book show us a glimpse of how to learn from the successes of the petrochemical industry. and systems thinking that incorporates topics such as land use and biodiversity in order to ensure a truly sustainable. While one of the Twelve Principles of Green Chemistry is devoted to ensuring that all feedstocks for both materials and energy are renewable rather than depleting. biofuels and biomaterials will be sustainable both for the planet as well as for profits.Foreword The development of the field of Green Chemistry has proceeded through a set of remarkable technological advances over nearly two decades. it is actually the case that the pursuit of the bio-based energy and material economy will rely on all of the principles of Green Chemistry and Green Engineering. As we begin thinking about the necessary transformation from essentially a petroleumonly based fuel system. The scientific and engineering breakthroughs contained in this volume are the essential building blocks that construct the foundation of this new technology platform. Biofuels need to pursue innovations that extract value from every element of the material and energy supplied by Nature. Through brilliant innovation. integrated material and energy flows. who sought to extract value from every aspect and distillation fraction of oil. The brilliant technologies discussed in this volume are an important step toward understanding biofuels within the integrated bio-based value chain. The oil business achieved this success not merely by identifying the fact that you can burn a black liquid that comes from the ground and get energy. . there are many who believe economic growth is inextricably linked to petroleum. The earliest stages of the oil industry were launched by visionaries such as Benjamin Silliman. resilient biofuel system. and the editors and authors are to be commended for constructing this high quality collection. In a matter of decades. This book provides an important review of the main issues and technologies that are essential to the future success of biofuels. Both are needed for success.

food production. By thoroughly understanding the inherent nature of the material and energy flows. While efficiency can be a good thing. Anastas New Haven. CT USA . we are more able to design a resilient sustainable system. In order to design. develop. their interaction with humans and the environment and their potential to cause adverse consequence anywhere in the life-cycle. it is far more important that a process is sustainable. and grow a viable biofuels future. climate change – efficiency alone will not get us on to a sustainable trajectory. This means understanding the long-term impacts of all aspects of the processes and the products on human health and the environment. implement. the biofuels world must also learn from the mistakes of the petrochemicals industry. there is a focus on how to make processes more efficient. water. resource depletion. ideally transformative innovations that move us to a more sustainable future. energy. When we look at the challenges of society and the world. Paul T. we must understand the fundamentals of sustainability. Innovations will be required. The technologies and perspectives in this book provide us with insight into some of these transformative innovations through Green Chemistry.xii Foreword However. Currently.

capital savings and depreciation are equal. The next wave of changes. As a result. and savings. Placed in this context of the Solow growth model. this model suggests the existence of an economic steady state where no additional growth can occur. Economic outputs are essentially the function of workforce size. depreciation. total factor productivity (TFP). Hans P. such as the so-called endogenous growth models. At the point of equilibrium. and consequently in a downwards displacement of the economic equilibrium point. A drawback of these latter growth models is that they do not account for the cost of sudden global disruptive technology changes (creation and adoption). Understanding these challenges that lie ahead is an important task to perform in order to design winning industrial strategies for the future. and that get integrated into the economy when capital returns. which acts by raising the rate of return per unit of capital employed. Verts. as the world economic engine integrates renewable energy technologies such as solar technologies or biofuels. and both capital and output are constant. and resources. to coal. other economic models that integrate the process of innovation creation. and revolutionary changes in energy cost and effectiveness. petroleum and nuclear technologies. have deeply shaped throughout history societal evolution worldwide. unless enabled by technological progress. a key component of the . sudden and durable large global economic disturbances thus result in a sudden and durable decrease in output. perhaps constitutes a greater challenge since predictably these technologies will be at least transiently less efficient than the conventional energies of today based on fossil and nuclear fuels. of course. The pressures exerted on the one hand by the cost of global waste treatment (such as atmospheric CO2 or methane) and by tensions in energy markets. all predict that continuous growth is achievable as long as capital is accumulated. with presumably a decrease in standards of living conditions. Blaschek and Hideaki Yukawa e Energy is a fundamental enabler of economy. thereby accelerating a rebound. for they integrate technological change as a continuous accumulation of R&D capital. which characterize such troubled times. whale oil. to be modulated by the increased appetite for risk and accumulation of latent innovation capital. capital invested.Preface Alain A. A simple model of economic growth was proposed by Robert Solow in 1956 where economic output per worker is calculated as a function of capital employed. This displacement typically results in improved living standards and conditions by maintaining on average gross domestic products (GDP) on trajectories of constant growth. which is accompanied de facto by a corresponding decrease in savings (such as R&D investments). Nasib Qureshi. On the other hand. This needs. and thus displaces upwards the equilibrium point. from animal and wood.

the European Union. the structure of the bioenergy business is analyzed through the lens of the manufacturers and end users. and methanol production from biomass. While. Biomass to Biofuels: Strategies for Global Industries. of fossil or nuclear fuel reserves. methane. but also by political interference leading to suboptimal choices as compared to marketbased choices. closing on global perspectives that exemplify paths towards resolving financing and commercializing hurdles of these innovative renewable energy and materials technologies. To this end. Consequently. Key milestones to be accomplished in each of these various enabling areas of the new energy and materials value chains are also defined with the aim to describe technological and technical transformation maps. various elements are reviewed here that describe the structure of the bioenergy business: a review of diesel. chemicals and materials. the characteristics of biofuels and renewable standards are described in major markets. The worldwide projected demand for biofuels is reviewed in light of public policies. and on the other hand by the cost of the adoption of new technologies on a global scale (such as renewable materials and sustainable energy) are likely to constitute a perfect storm for the global economy. or by the burst of economic bubbles that have a direct negative impact on investments. First. in conditions of limited resources. In a context of constant population expansion. and for delaying or decreasing today’s CO2 remediation expenditures. as well as potential opportunities for new jobs and new products creation. in the absence of perfectly equivalent substitutes there is thus a strong preference (akin to a high discount rate) for the value (and use) today. capital investments for technological innovation may extend the useful life of finite reserves or introduce resource substitutability properties. GDP per capita. and energy use per unit of GDP. instead of tomorrow. hydrogen. Biofuel demand realization is explored by analyzing . beyond suggesting that the exhaustion of energy sources would obviously stall the economic engine.xiv Preface resource parameter of the economic function. Derivation of the economic function applied to energy suggests that the variation of the energy need is the sum of the variations affecting population. This can even be exacerbated not only by resistance to change originating from an array of vested interests. with particular consideration given to policies in Brazil. thus generating a sense of urgency. with a particular emphasis on the various chemical attributes of biodiesel (ethyl/methyl esters) and bioethanol. this time horizon expansion of finite reserves is dependent on choices (akin to discount rates used in corporate finance) made regarding utilization rates and energy efficiencies. Japan and the US. unless near term environmental and economic threats of dramatic consequences can be readily identified and acknowledged. as exemplified by the housing asset crisis that occurred during the last few years of the first decade of the 21st century. combining science and business perspectives pertaining to innovation creation and adoption of innovative technologies in the field of sustainable energy is a critical task to accomplish for Society as a whole to efficiently cope with the current period of transition from fossil fuel-derived energy. chemicals and materials to renewable energy. As demonstrated by the controversy surrounding the Kyoto protocol and subsequent protocols to regulate CO2 emissions. ethanol and butanol methods of production from biomass. In Part I of the present monograph. this equation suggests that any change that tends to decrease energy effectiveness (such as a dramatic rise in energy cost due to energy supply disequilibria or to the integration of the cost of atmospheric CO2 or methane remediation) would negatively impact the economic output if such a rise is not offset by a corresponding increase in TFP.

with a particular assessment of the biodiesel and ethanol volumes that could be sustainably generated using all these different raw materials and using existing arable lands. This section closes with considerations regarding under which circumstances a biorefinery to generate an array of useful products from a variety of primary raw materials is preferable to a dedicated manufacturing plant. the question is addressed whether generic pretreatments exist that can be applied to most biomass sources. conventional biotechnological ethanol and butanol production technologies are reported together with their newest advances. and notably the use of ethanologenic gram(-) bacteria that can catabolize a large spectrum of sugars. an array of advanced fermentation technologies are assessed such as fed-batch. What is more. fermentation and downstream processing technologies are both considered here. The use of alternative microbial converters to produce ethanol is also reviewed. A chapter on the thermo-chemical pretreatments of lignocellulose serves as a brief introduction to these industrial steps in biomass processing and to the reasons why such pretreatments still remain inescapable. and extraction. for example by converting a greater share of the energy value of the biomass into ethanol. The purpose of the chapter is to identify opportunities of technological development to render ethanol production more cost-effective. and cell recycle membrane systems. Advanced technologies for biomass hydrolysis and saccharification are subsequently reported. or combined chemical and enzymatic treatments. for fuel. These perspectives include the liquefaction and gasification of solid biomass. the various technologies required to produce diesel from biomass are reviewed to provide a snap-shot of the present-day technology and a view of the future. . as compared to existing industrial fermentation processes. stripping. immobilized cell. continuous. the use of vegetable oils in transesterification reactions to yield biodiesel. Notably. Emphasis is placed on the desirable traits of these pretreatment processes and on their relative comparative advantages. Commonly encountered biomass hydrolysis inhibitors are subsequently described. and the need to better preserve Nature’s environment capital. ammonia. The discussion around downstream processing presents alternative product recovery technologies including adsorption.Preface xv the global biofuel production potential using sugars such as sucrose or hexoses and lignocellulosic materials. and the production of algal oil as biodiesel raw materials. growth-arrested cells at very high cell densities. The first chapter of this section thus presents a review of the economics and technologies of current industrial processes for ethanol production using baker’s yeast as a natural ethanol producer. In Part III. together with methods to achieve their removal from fermentation media. sodium hydroxide. pervaporation. Notably. including treatments that make use of dilute sulfuric acid. Several of the advantages of these novel systems. chemicals or biomaterials. or whether tailor-designed pretreatments are needed. the Fischer-Tropsch process to generate diesel from syngas. In Part II. The discussion includes economic considerations as emphasis at both the laboratory and industrial scale must be placed not on optimizing biological performance. are discussed in detail. This particular chapter is completed by a description of the conversion of lignocellulosic materials into bioethanol using recombinant yeasts. as well as hurdles to their industrial-scale implementation. This analysis includes a detailed comparison of the advantages and drawbacks of each of these technologies. as well as the use of bacteria that exhibit intrinsically high industrial robustness such as Lactobacilli or Corynebacteria. An important theme is of course that of the tension between the use of agricultural commodity for food vs. but rather on optimizing the performance and rewards of the complete ethanol or butanol value chain.

or perhaps to a greater extent solar technologies) would become the dominant form of energy utilized by the end user. hydroelectric. a brief period of transition. Methanol production and utilization methods are also presented in a brief overview in the final chapter of this section. geothermal). including for transportation. that will in the long run enable the displacement and replacement of the combustion engine and its associated value chains. by a superior technology (the electric engine) and associated value chains. in principle constitutes. Of course. This chapter comprises financing of large scale manufacturing projects (such as biorefineries and multi-million gallons dedicated plants). photovoltaic. In Part IV. methane and methanol production technologies are described. Part III closes on the use of clostridia as fuel producers. since such technologies could be critical to implement alternative (e. with a brief description of the use of this compound as a replacement fuel and of its challenges. financing strategies for very large-scale biofuel production and technology development start-ups are described to better mitigate financing hurdles in the domain of bioenergy. to a more decentralized model where electricity (produced from a variety of means including increasingly from renewable sources. as opposed to fuels.xvi Preface Emphasis is placed on technology development for the cost-effective recovery from dilute streams. Also. technologies for the industrial scale production of methane are reviewed in the following chapter. yet measured in a period of a decade or more. and financing of biofuel technology start-ups including the crucial role that composition of matter patents protecting the production and sale of novel materials are likely to play in the expansion of a renewed chemical industry. hydrogen. electricity.. Axes of development in chemical and process engineering for converting biomass to energy are discussed with the aim to provide a synthesis of the portfolio of technologies that were described in the preceding chapters. A key feature of these latter two chapters is to set the goals that must be achieved in this arena in order to manufacture and use hydrogen on a costcompetitive basis. during which the world will move from a centralized model of energy production with a diversified energy mix (electricity. such as Aeolian. with all its related economic benefits. hydroelectric. fossil fuels). This forward looking chapter stems from the needs of the biofuel industry to establish a few concrete possible technical solutions that could be implemented directly into the agricultural fields with the view to minimize changes to agricultural practice and to maximize public acceptance over the large scale planting of enhanced energy crops. Part V constitutes a perspectives section to highlight two critical hurdles to the realization of the biofuels vision: the genetic engineering of biomass-producing crops and the financing of the new industry. including smart distribution grids. beyond a detailed review of the science of bioenergy and replacement transportation fuels. a universal energy currency and it can be mass produced cheaply and essentially ubiquitously. bacterial) production systems. The key messages of the monograph. It is this fundamental feature. including particularly the generation of hydrogen by microbial cultures and by undefined consortia of microorganisms. including the latest technological developments and the relevant economic modeling of the acetonebutanol-ethanol fermentation.g. In addition. is that perhaps the period of transition that one is currently witnessing is maybe just that.g. Indeed. The first chapter of Part V thus provides a description of plant engineering techniques to enhance biofuels primary raw materials. given its very low entropy. the contribution of forms of renewable energies other than those derived from biomass are summarized here (e. Aeolian. this in . Finally..

India.Preface xvii itself constitutes a leap of faith. since these technologies and their deployment at the industrial scale would become useful to drive the next wave of transformation of the petrochemical industry via the creation of novel industrial materials and renewable commodity chemicals markets. photovoltaic and thermal technologies to harness. butanol. and chiefly for transportation purposes. several automobile makers are already embracing this challenge. managing the period of transition is critical to maintain global trends of economic growth. and restitute at will solar energy. methane or methanol would nevertheless not have been in vain. ethanol. store. as the appropriate electric batteries to store and restore cheaply and efficiently their energy contents remain to be developed. Should this vision become true in the long-term. and the deployment on a global scale of biofuels. including in regions of high growth potential such as the BRIC countries (Brazil. China) or countries of the African continent. the technologies and value chains developed to produce biotechnological diesel. for example. Notably. Russia. . represents an important link and a crucial complement to the continued development and deployment of. What is more. hydrogen.


Eggenstein-Leopoldshafen. Rehovot. Center for Advanced BioEnergy Research. Microbial Ecology Group. Israel John Benemann. USA Nicolaus Dahmen. Ezeji. South Dakota State University. Germany Hans P. Flint. Institute for Genomic Biology. Dolan. SD. Institute for Technical Chemistry. Plant Biochemistry. Lund University. Biology/Microbiology Department. CA. Urbana. Brulc. USA B€rbel Hahn-H€gerdal. Wooster. USDA. Faculty of Biology & Biotechnology. Walnut Creek. Germany Bruce Dien. ARS. Urbana. Aberdeen. USA Thaddeus C. Peoria. USA William Gibbons. Bochum. Institute for Genomic Biology. Department of Applied Microbiology. Weizmann Institute of Science. IL. USA Harry J. University of Aberdeen. UK Jon Van Gerpen. University of Illinois at Urbana-Champaign. Forschungszentrum Karlsruhe. Department of Food Science & Human Nutrition and The Institute for Genomic Biology. USA Margret E. Sweden . Urbana. Rowett Institute of Nutrition and Health. IL. USA Gregory A. Moscow. Department of Animal Sciences. Bayer. USA Gbor Bernt. Benemann Associates. University of Idaho. Department of Biological and Agricultural Engineering. Department of Animal Sciences. VA.Contributors Edward A. Ruhr University a a Bochum. IL. a a Lund. National Center for Agricultural Utilization Research. USA Jennifer M. Department of Animal Sciences and Ohio State Agricultural Research and Development Center. Blaschek. Brookings. IL. OH. Methanol Institute. The Ohio State University. University of Illinois at Urbana-Champaign. Department of Biological Chemistry. University of Illinois at Urbana-Champaign. Arlington. Berg Miller.

Gokasho. Pacific Northwest National Laboratory. Peoria. Eggenstein-Leopoldshafen. Urbana. RISH. P. Department of Agricultural and Biological Engineering. Israel Chia fon F. Germany Dimitrios C. Agricultural Research Service. Lee. United States Department of Agriculture. Ramat Aviv. Tel Aviv University. Nagoya. Bioproducts and Biocatalysis Unit. Institute for Technical Chemistry. Plant Biochemistry. Agricultural and Biological Engineering and Ohio State Agricultural Research and Development Center. Plant Biochemistry. IL. USA Masayuki Inui. University of Illinois at Urbana-Champaign. Bioproducts and Biocatalysis Unit. Mizuho. Faculty of Biology & Biotechnology. Graduate School of Natural Science. KS. University of Illinois at Urbana-Champaign. Germany Takahisa Hayashi. Chinese Academy of Sciences. Wooster. Bochum. Wyndmoor. Faculty of Biology & Biotechnology. Urbana. China Siqing Liu. Department of Food. Eggenstein-Leopoldshafen. Hansen. USA Raphael Lamed. Uji. ICM. USA Andrea Kruse. Molecular Microbiology and Biotechnology Group.xx Contributors Alan C. Kyoto. Germany Edmund Henrich. Kyoto University. Inc. Japan Anja Hemschemeier. United States Department of Agriculture. Ruhr University Bochum. USA Rumi Kaida. National Center for Agricultural Utilization Research. Department of Mechanical Science and Engineering. Research Institute of Innovative Technology for the Earth (RITE). Japan Shin-ichiro Kidou. USA . Ruhr University Bochum. Kyoto University. WA. University of Illinois at Urbana-Champaign. RISH. IL. USA Yebo Li. Institute for Technical Chemistry. Gokasho. Kyritsis. Department of Molecular Microbiology and Biotechnology. The Ohio State University. Nagoya City University. IL. Kyoto. Yamanohata. State Key Laboratory of Coal Conversion. Marine Sciences Laboratory. IL. Shanxi. Kyoto. Urbana. Eastern Regional Research Center. Japan David B. Peoria. USA Yong-Wang Li. Sequim. OH. Forschungszentrum Karlsruhe. Colwich. Institute of Coal Chemistry. Taiyuan. Forschungszentrum Karlsruhe. USA Thomas Happe. National Center for Agricultural Utilization Research.. Bochum. Johnston. IL. Hughes. Japan Scott Kohl. Uji. United States Department of Agriculture. USA Stephen R. R. Germany Michael Huesemann. Department of Mechanical Science and Engineering.

USA Vijay Singh. Japan Nasib Qureshi. USA . Bioenergy Research. National Institute of Advanced Industrial Science and Technology. E. Faculty of Biology & Biotechnology. USA Grant Stanley. Universit€t Hamburg. The Ohio Agricultural Development and Research Center. Plant Biochemistry. Melbourne. Victoria University. United States Department of Agriculture. USA J€rgen Scheffran. Germany Nobuyuki Nishikubo. University of Illinois at Urbana-Champaign. WA. USA Katrin M€llner. The Ohio State University. Department of Agricultural and Biological Engineering. OH. National Institute of Advanced Industrial Science and Technology. Tumbleson. Australia M. Lewis Liu. Tsukuba. USA Klaus Raffelt. Japan Masaru Ohme-Takagi. Bochum. School of Molecular Sciences. Marine Sciences Laboratory. Rausch. Peoria. Department of Animal Sciences. Urbana. Bioenergy Research. IL. Formerly based at the Center for Advanced BioEnergy Research and the Energy Biosciences Institute. USA G. Germany Floyd L. Faculty of Biology & Biotechnology. Ruhr o University Bochum. Institute for Geography. Research Institute of Genomebased Biofactory.Contributors xxi Z. IL. Department of Agricultural and Biological Engineering. Forschungszentrum Karlsruhe. Research Institute of Genomebased Biofactory. Gene Regulation Research Group. Urbana. USA Matthias R€gner. Sequim. National Center for Agricultural Utilization Research. The Ohio State University. Department of Agricultural and Biological Engineering. Ruhr u University Bochum. Yokohama. USA Nobutaka Mitsuda. University of Illinois at Urbana-Champaign. Urbana. University of Illinois. IL. IL. Ruhr University u Bochum. Germany Thilo R€hle. Columbus OH. Faculty of Biology & Biotechnology. Richland. Institute for Technical Chemistry. Peoria. Plant Biochemistry. National Center for Agricultural Utilization Research. Plant Biochemistry. KlimaCampus. Wooster. Schanbacher. IL. WA. United States Department of Agriculture. Urbana. Gene Regulation Research Group. IL. Pacific Northwest National Laboratory. Department of Animal Sciences and Environmental Science Graduate Program. Bochum. Tsukuba. USA F. u a Germany. Roesjadi. Blaine Metting. Pacific Northwest National Laboratory. Bochum. Japan Mark Morrison. Germany Kent D. RIKEN Plant Science Center. Eggenstein-Leopoldshafen. University of Illinois at Urbana-Champaign. The Ohio Agricultural Development and Research Center.

Institute of Coal Chemistry. Ashkelon. Israel Kouki Yoshida. The Ohio State University. London. USA Hideaki Yukawa. Ruhr University Bochum. Kyoto. Chinese Academy of Sciences. Japan Zhongtang Yu. USA Jian Xu. London Business School. Plant Biochemistry. Department of Animal Sciences and Environmental Science Graduate Program. ATI Technological Incubator. Zomet Abba Hillel. Institute of Coal Chemistry. UK e Nadine Waschewski. Technology Center.xxii Contributors Alain A. IL. China Yong Yang. Taiyuan. Bochum. Faculty of Biology & Biotechnology. Shanxi. Research Institute of Innovative Technology for the Earth (RITE). Shanxi. Germany Bryan A. White. Verts. Yokohama. State Key Laboratory of Coal Conversion. R. Columbus. Totsuka-ku. University of Illinois at Urbana-Champaign. P. China Sarit Soccary Ben Yochanan. R. Taiyuan. Japan . Taisei Corporation. Urbana. Departments of Animal Sciences and Pathobiology. Ashkelon High-Tech Park. The Ohio Agricultural Development and Research Center. The Institute for Genomic Biology. Ohio. Chinese Academy of Sciences. Molecular Microbiology and Biotechnology Group. P. Sloan Fellowship. State Key Laboratory of Coal Conversion.

Part I Structure of the Bioenergy Business .


For example. hydrogen derived from biomass is considered as the ideal fuel. a greater diversity of primary raw materials for manufacturing renewable transportation fuels will be used. including an array of recycled materials. lignin. Lee 1.1 Characteristics of Biofuels and Renewable Fuel Standards Alan C. the conversion of biomass to liquid fuel via pyrolysis is receiving attention. ethanol production from cellulosic material is likely. is being explored as an alternative [1]. waste oils. Nasib Qureshi. Biomass to Biofuels: Strategies for Global Industries and Hideaki Yukawa Ó 2010 John Wiley & Sons. or virgin oils processed either pure or blended with crude oil using petroleum refinery or similar operations. However. there are several technical hurdles that will need to be circumvented before this vision becomes reality. It is expected that. including not only the production of hydrogen from renewable materials but also safe methods for the storage and transport of hydrogen fuels [2]. or triglycerides. Furthermore.1 Introduction Liquid biofuels currently in commercial use comprise primarily ethanol-derived fuels. Hans Blaschek e . Kyritsis and Chia fon F. its use as a biofuel for transportation remains marginal to this date. mainly from grain. the use of hydrogenation-derived renewable diesel and gasoline from fats. Hansen. Ltd Edited by Alain Verts. as well as butanol production from grain and possibly also from cellulose. sugarcane or sugar beet. as well as the production of alkanes from the hydrogenation of carbohydrates. in the future. Although methane production from waste materials is already well established. In the long term. because its combustion yields zero carbon dioxide. and biodiesel produced from a variety of vegetable oils and animal fats. In addition. Dimitrios C.

Acid catalysts may also be used. although other biofuels will also be mentioned when comparing the key properties of these materials. Alcohols are defined by the presence of a hydroxyl group (–OH) attached to one of the carbon atoms. with sodium hydroxide being more commonly used because of its availability as a drain-cleaning chemical. they may be a mixture of typically five to eight esters of fatty acids. A primary factor that distinguishes fuel alcohols and biodiesel from petroleum-based fuels is the presence of oxygen bound in the molecular structure. the relative composition of which is dependent on the raw material source. methane or ethanol.1 Transesterification reaction to produce biodiesel (R represents a primary alkyl radical). biofuels may consist of pure single-component substances such as hydrogen. Butanol is a more complex alcohol than ethanol as the carbon atoms can form either a straight-chain or a branched structure. alternatively. While straight vegetable oils have been used to power diesel engines. the use of these homogeneous catalysts involves process steps for the removal of FFAs and water from the feedstock and catalyst from the products. the choice being dependent on the amount of free fatty acids (FFA) in the raw oil [3].1. as in the case of biodiesel. as conventional engines have not been designed to be operated with relatively viscous fluids. Butanol production from biomass tends to yield mainly straight-chain molecules. their viscosity is much greater than that of conventional diesel fuel. 1. Typical alkali catalysts are sodium hydroxide and potassium hydroxide. This is an important difference. such as zeolites [4]. It is worth noting that although ethanol (creating ethyl esters) and CH2-OOCR | CH-OOCR + 3 CH3-OH | CH2-OOCR Catalyst → 3 CH3-OOCR + CH2OH | CHOH | CH2OH Glycerol Vegetable oil Alcohol Vegetable oil (Triacylglycerol) Alkyl ester (Biodiesel) Figure 1. Apart from corrosion problems. petroleum-based fuels are a blend of a very large number of different hydrocarbons. the molecular structure of ethanol is C2H5OH. the characteristics of biofuels will be focused primarily on ethanol and biodiesel. An alternative being explored is that of heterogeneous nanocatalysts. thus creating methyl esters. as shown in Figure 1.4 Structure of the Bioenergy Business In this chapter. these biofuels are typically blended with petroleum-based fuels. In order to reduce the viscosity. In addition. This relatively finite number of fatty acid esters in biodiesel contrasts with the much broader and more complex range of hydrocarbons that exists in petroleum. and hence problems may be encountered when fuel vegetable oil is injected into the engine. and that of butanol is C4H9OH. For example. that eliminate steps in conventional biodiesel production .2 Molecular Structure Although in general. The alcohol typically used in the reaction is methanol. one widely used method is to transesterify the vegetable oil or animal fat via a chemical reaction between the oil or fat and a mixture of an alcohol and a catalyst. thus resulting in different properties.

Vegetable oils and animal fats consist of a mixture of fatty acids with carbon chains of different lengths as well as different degrees of unsaturation (i. this results in the fuel gelling or solidifying at higher temperatures than would diesel fuel. A comparison of the . and one double bond [6] other alcohols can also be used..Characteristics of Biofuels and Renewable Fuel Standards 5 Figure 1. while increasing the degree of unsaturation causes the cetane number rapidly to drop. C18:1 denotes that there are 18 carbon atoms in the chain.2 Fatty acid profiles of five biodiesel source materials. the process requirements are different when using these latter feedstocks. engine manufacturers design their products based on expectations regarding the properties of the fuel to be used by the consumer. This need for retrofit explains in part the resistance initially displayed by various automobile manufacturers to implement the use of ethanol.3 Physical Properties To a significant extent. the value of a higher cetane number with a longer carbon chain length is offset by increasing cloud and pour points. Figure 1. The introduction of biofuels such as ethanol and biodiesel has naturally led not only to a closer scrutiny of the standards of these new fuels. In turn. an increase in chain length leads to a higher propensity for the fuel to selfignite under conditions of high heat and pressure (this property is measured by the cetane number). this information is used to modify engine control parameters in order to account for changes in fuel properties that could affect fuel atomization and combustion should the engine remained unchanged. the number of double bonds that may exist in the fuel molecule). These factors are discussed later in the chapter. However. 1.e. For example.2 shows the fatty acid ester composition of selected source materials for producing biodiesel. Both the chain length and the degree of unsaturation have a major effect on the fuel properties of the esters in the biodiesel [5]. but also to attempts at characterizing their properties in detail.

The volatility of a fuel is determined by the Reid Vapor Pressure (RVP). The mixing process relies on sufficient atomization of the fuel and its dispersion to occur either at the level of the intake of the engine or in the combustion chamber. which is five orders of magnitude higher than the one of hydrocarbons [20–23]. and then chemically reacted and burnt. Although ethanol has a much lower RVP than gasoline. Mixing of the fuel and air is achieved as these two components enter the cylinder. as the cost of fuel ethanol production – particularly at the downstream purification step – is dramatically reduced when this specification is allowed. ignite. such relatively large amounts of water could not be accommodated in typical ethanol–gasoline blends as the ethanol would separate from the gasoline. In the United States and other countries. the fuel is injected into the inlet port immediately before the intake valve during the intake stroke. In this context. The ability of the SI engine fuel to vaporize at engine start-up when cold is another very important characteristic.6 Structure of the Bioenergy Business key properties of biofuels and petroleum-derived fuels is provided in Table 1. This is important. For example. this difference results in the biodiesel . which in turn vary with the biomass raw material from which these fuels derive (see Figure 1. as well as to prevent the occurrence of vapor locks in the fuel systems.1) [15]. as a result. the fuel atomization process is critical as there is very little time for the fuel to be injected into the combustion chamber. Notably. with a maximum being reached at about 10 % ethanol. (ii) viscosity. or cruising. vaporized. Direct injection into the combustion chamber has also been introduced as a means to permit a much leaner overall combustion process. This is the primary reason why a blending limit of 75–85 % ethanol (E85) is specified by regulators. The physical characteristics that affect the atomization and fuel–air mixing process include: (i) fuel density. Ethanol has a much higher latent heat of vaporization than gasoline fuel (see Table 1. As compared to conventional diesel. these engines either rely on gasoline being provided for cold starting. as well as how it performs initially during vehicle idling. and (iii) surface tension. its ability to combust. The physical properties of the fuel influence how well the fuel mixes with air and. or they may use a specifically designed process for heating the fuel and the air in the manifold before the engine is started.1. the RVP is regulated for both conventional gasoline and ethanol blends to reduce evaporative emissions. there have been recent indications that electrostatically assisted atomization may become a useful technology because of the relatively high electric conductivity of ethanol. both processes require the fuel to be sufficiently volatile to facilitate mixing. in order to ensure that there is a sufficient amount of an adequately volatile gasoline to vaporize. which means that more heat is required to cause fuel ethanol to vaporize as compared to conventional gasoline. engines have been developed and used in Brazil and Europe that run on E100. as it determines the ease with which the engine will start. and thus allow the engine to start when cold. the density of biodiesel is approximately 7 % higher than that of diesel fuel. One major advantage of combustion engines that run on E100 is their ability to tolerate the presence of up to 5 % water in the ethanol fuel. biodiesel typically exhibits higher values for all of the characteristics listed above. mixed with air.1). mix with the air. Notably. before switching to ethanol once the engine has warmed up. For most spark ignition (SI) engines. it is well known that the addition of ethanol to gasoline raises the RVP. however. acceleration. For diesel engines. For example. a single digit measure of a fuel’s propensity to evaporate. These properties are strongly influenced by the fatty acid profiles of the biodiesel fuels.

8 46.5 Characteristics of Biofuels and Renewable Fuel Standards a (A/F)s: Stoichiometric air/fuel ratio. BD: Biodiesel.8 37–39.5 12.9 49.9 33.6 0 0 0 10.5 12.7 12.0 49.9 39.2 15.1 38. 7 .7–44. The values of these properties were assembled from data in Refs [7–19].7 12.3 350–356 507 — 428 423 1103–1186 842 585 — 270–286 230 320 320 325 320 313 325 313 48–103 — — — 1434 32 22 2.4 43.8 15.9 10.7 6.1 34.5 12.8 9.9 44. Octane rating is also known as antiknock index or octane number.6 17.3 40.8 38. LHV: Lower heating value. LPG: Liquefied petroleum gas.0 34. RVP: Reid vapor pressure.5 9.9 26.4 12.2 12.4 11.6 49 50–62 51.2 58 Latent heat of vaporization (kJ kgÀ1) RVP (kPa) Cetane rating Octane rating RON 91–99 130 > 120 104 112 106–112 107–111 96 n.8 21. Cetane Rating is an estimation of ignition quality.3 11.1 Biofuel properties compared to petroleum-based fuels (A/F)s Oxygen content (% by mass) 0 0 0 0 0 50.a.0 46.5 14. — — — — — — — — — MON 82–90 130 120–127 89 97 91–92 89–92 78 n.8 11.1 119.2–51 52.2 16.6 37.a. — — — — — — — — — Fuel LHV (MJ kgÀ1) Gasoline Methane Natural gas LPG Propane Methanol Ethanol 1-Butanol Hydrogen Light diesel Heavy diesel Soybean BD Rapeseed BD Canola BD Sunflower BD Palm BD Cottonseed BD Tallow BD 42.0 11.2 42.1–38.4 11.8 37.Table 1.9 14.2 19.3 — — — — — — — — — — — — — — — 3 8 25 — 40–65 40–65 46.3 14.

when it is compared per unit mass – as used in the energy equation – the heat capacity of biodiesel is lower than that of diesel. which would be expected to have a significant influence on the spray evaporation process. it is expected that the evaporation of the biodiesel would be less efficient compared to petroleum-derived diesel fuel at those high temperatures Surface tension is one of the most important properties in spray breakup and collision/ coalescence models. the heat capacity of biodiesel per unit mole is almost 50 % higher at temperatures near these fuels’ boiling points. the vapor pressure of biodiesel is much lower. limit ASTM D975 @ 40°C 4 3 2 1 0 20 30 40 50 60 70 80 90 100 Temperature.3 Variation with temperature of measured kinematic viscosity of biodiesel made from five source materials. and wall film motion. the liquid viscosity of biodiesel is higher than that of diesel. Consequently. °C Figure 1. Therefore. mm^2/s 7 6 5 Max. compared to that of diesel fuel.3 illustrates the differences in viscosity of biodiesel fuel made from the five raw materials shown in 9 Soybean 8 Rapeseed Coconut Palm Beef Lard #2 Diesel Kinematic Viscosity. 2 diesel fuel and to the ASTM standard D975 for diesel fuel [6] . it is to be expected that the atomization process will be affected by the viscosity difference. Empirical correlations have suggested that surface tension is a linear function of temperature. and thus becomes significantly higher than that of diesel at higher temperatures. the heat of vaporization of biodiesel is lower at low temperatures. Compared to diesel. compared to no. Likewise. Figure 1. Furthermore. at room temperature the surface tension of biodiesel is about the same as that of diesel. Liquid viscosity is an important parameter with regard to droplet atomization. drop internal flow. especially at low temperatures where most of the atomization processes take place during the injection process in the engine. but it becomes higher at high temperatures. relative to conventional diesel the surface tension decreases more slowly with increasing temperatures.8 Structure of the Bioenergy Business droplets penetrating deeper into the combustion chamber because of their higher momentum when injected. Notably. However. and therefore that it is relatively independent of the specific methyl ester mix that composes different varieties of biodiesel [6]. However. This suggests that biodiesel droplets are heated faster than diesel droplets. As the spray droplets are heated quickly during the vaporization process in the engine.

surface tension and thermal conductivity between diesel and biodiesel fuels. the diffusivity for biodiesel vapor is much lower than that for diesel by as much as a factor of 20. Notably.Characteristics of Biofuels and Renewable Fuel Standards 9 Figure 1. Exploitation of these results provide a guideline on the desirable characteristics of blended fuels. Typically. (v) liquid thermal conductivity. 18] included: (i) liquid density. the most impactful of these characteristics seem to be liquid fuel density. while its thermal conductivity is about 30 % lower than that of diesel [24]. Due to its lower boiling point and critical temperature. which in turn affects the transient heat transfer from the surrounding gas mixture to the drop surface. and lard biodiesels have been predicted. It is particularly noteworthy that these properties differ considerably between each of the biodiesel fuels analyzed. The viscosity of biodiesel vapor is about 60 % higher than that of diesel. This latter observation can perhaps be ascribed to the importance of these parameters on the atomization. coconut biodiesel shows a tendency to vaporize faster than any of the other . Recent studies using multidimensional spray and combustion modeling have been conducted to investigate the effects of varying the fuel’s physical properties on the spray and combustion characteristics of diesel-engines when these are operated using various biodiesel fuels [17. 18. The properties of typical biodiesel fuels that have been used in these studies. As described earlier. (vii) latent heat. these observations support the view that there is no single physical property that dominates the differences of ignition delay between diesel and biodiesel fuels. (ii) vapor pressure. The results suggest that the intrinsic physical properties of each of these fuels significantly impact spray structure. However. 25–28]. (iii) surface tension. relies heavily on the physical properties of the fuels being analyzed. The sensitivity of the computational results to individual physical properties is also investigated by changing one property at a time.2). the physical properties and critical temperature of soybean. and mixture preparation processes. are compared with those of conventional diesel fuels. and thermal conductivity – can all be estimated for biodiesel mixtures. Moreover. and the simulation results obtained. spray. The transport properties of the vapor phase – that is. diffusivity. This is particularly important when the fuel drops rapidly vaporize so that the fuel–air mixture become richer. ignition delay and burning rates in a wide range of engine operating conditions. and (xi) vapor thermal conductivity. the thermal conductivity of the biodiesel is slightly lower than that of the diesel [6]. The properties investigated in Refs [17. as compared to diesel fuel. viscosity. The vapor heat capacity of the fuel is an important parameter that influences the thermal energy balance and temperature distribution of gas mixtures surrounding the spray drops. (vi) liquid specific heat. among the biodiesel fuels there is substantial variation caused by differences in fatty acid content. viscosity. and surface tension. vapor pressure. Moreover. however. palm. and which is used to model the breakup of fuels in diesel engines. (iv) liquid viscosity. (viii) vapor specific heat. (x) vapor viscosity. The vapor heat capacity of biodiesel is slightly lower than that of the diesel [6]. (ix) vapor diffusion coefficient. Remarkably. coconut. Using this model and the fatty acid profiles of the source oils for biodiesel (as shown in Figure 1. Liquid thermal conductivity affects the heat transfer between the drop interior and the surface. all of these biodiesel fuels have higher viscosities. a recent study has shown the effect that these differences have on fuel vaporization [6]. significant differences exist in density. The spray atomization model thus developed.2.

Note also that the energy content of butanol is much greater than that of ethanol. as the blend percentage increases to more than 5 %. Such a property is represented by the cloud point and cold filter plugging point (CFPP). This property is strongly affected by the fatty acid profile of the biodiesel. and is influenced by the relative proportion of saturated and unsaturated fatty acids. this makes them susceptible to clogging of the fuel system and preventing the engine being started when cold.1 illustrate the variation in energy content for a range of fuels. simulations for the biodiesel blends predict vaporization similar to that of diesel fuel. Likewise. some hydrocarbons in diesel fuel begin to solidify. making it an attractive alternative fuel for SI engines. B5 and B20 have also been demonstrated. the fuel vapor mass is shown to decrease.4 Chemical Properties The chemical properties of biofuels are strongly affected by their different molecular structures. Ethanol has about a 30 % lower energy content than gasoline on a per unit volume basis. thus making the mixture leaner. this translates into a lower distance traveled per tank of fuel compared to gasoline. The presence of oxygen in the molecule (see Table 1. At low blend percentages. These blends were modeled using the spray code including multicomponent fuel effects [17. Nonetheless. Significant differences in the spray and vaporization between diesel–biodiesel blends of B2 (2 % biodiesel: 98 % diesel). for which biodiesel fuels generally have higher temperatures. The diesel fuel blends have a lower overall spray tip penetration than pure biodiesel. Energy density is a measure of how much energy a fuel contains. Here. 44–47]. this characteristic is partially due to differences in molecular weights and densities of the fuels. as described earlier in the chapter. Computed spray structures also demonstrate a relationship between atomized droplet diameter and fuel vaporization. The lower heating values (LHV) reported in Table 1. 29–43]. The vapor mass composition is also affected by the blend percentage and lower volatility of biodiesel. thereby inhibiting the flow of fuel from the storage tank to the fuel injection pump via the filter system. additives such as malan-styrene esters and polymethacrylate have been used successfully to address this limitation [19. However. such as B2 and B5. In the case of gasoline engines – which must run with an air–fuel mixture that is consistently close to the chemically correct or stoichiometric ratio to achieve complete combustion – the addition of ethanol to gasoline results in there being extra oxygen in the combustion chamber contributed from the ethanol. The biodiesel fuels that behave most like pure conventional diesel include palm and lard biodiesel. this characteristic has a direct impact on how much power an engine produces by combusting this particular fuel. The addition of 10–15 % ethanol to gasoline is widely . the higher the cloud point and CFPP temperature. biodiesel exhibits an energy content that is approximately 9 % lower than that of conventional diesel. The higher the proportion of the saturated component. 1.10 Structure of the Bioenergy Business pure biodiesel fuels when injected under engine-like conditions. Another important characteristic of diesel fuels is their ability to retain liquid properties under cold weather conditions.1) naturally causes a leaner combustion process in existing engines because this oxygen also participates in the combustion process. when ambient temperatures are low enough. On the other hand. the difference on a volume basis is lower because the higher density of biodiesel compared to standard diesel fuel helps to offset the difference in energy content.

and have been identified in the early studies conducted by Norton and Dryer [51]. 1. The octane number is an important parameter that establishes whether or not a fuel will knock in a given SI engine under given operating conditions.1). This is an important characteristic.4.4. One of the reasons for the higher octane rating exhibited by ethanol is the relatively high heat of vaporization that characterizes this compound (Table 1. summarized in the classical text authored by Glassman [49]. In turn. In a similar way. the cetane number (or cetane rating) provides a measure of compression ignition. In addition.Characteristics of Biofuels and Renewable Fuel Standards 11 practiced. The main variations that occur with respect to the combustion of hydrocarbons are due to the presence of a hydroxy group.1 Alcohols: Ethanol The combustion chemistry of light alcohols has been studied extensively. this in turn increases the engine overall efficiency and offsets to some extent the lower energy content of ethanol. However.1 have higher cetane ratings than those of the diesel fuels available in the US (about 43). engines designed to run on E100 use a comparatively higher compression ratio. One very attractive chemical property of ethanol is its resistance to self-ignition. a higher octane number also means that an engine can burn ethanol at a higher compression ratio. as reflected by its high octane number (see Table 1. as gasoline engines are able to tolerate such volumes of ethanol in gasoline with only small changes to the air–fuel ratio. as a result. Here. where the consumers have a large choice (63 different models in 2007) and the market penetration of this type of automobiles is high.1 Oxidation and Combustion Chemistry Biological processes can yield light fuel molecules such as bio-hydrogen or bio-methane. but they have ratings comparable to the ratings of the diesels available in Europe (about 50). as demonstrated by a total of 85. in modern engines a sensor in the exhaust detects changes in the mixture and provides feedback to the system that controls the mixture ratio. Most of the biodiesel fuels listed in Table 1. The higher the octane number of a fuel. consequently. 50]. the combustion chemistry of which is well established. the higher resistance it has to knock.6 % of the new automobiles sold in 2007 in that country [48]. with a particular emphasis on the combustion of alcohols and fatty acid esters. which in turn slows down the combustion and provides a higher resistance to knock. and reviewed more recently in a more brief form by Law [50]. ethanol can be used as an octane enhancer for gasoline. Consequently. the greater the ignition quality of a fuel and the shorter the ignition delay.1. A higher heat of vaporization results in cooler fuel–air mixtures. The higher the cetane number is. Especially for ethanol. since long ignition delays result in most of the fuel being injected before ignition occurs. a blend discussed earlier. diesel knock can occur.1). we focus instead on the chemical issues associated with the combustion of heavier biofuels.4 (adapted from . this results in very fast burning rates and very high rates of pressure rise once ignition starts such that. These flex-fuel vehicles are particularly common in Brazil for example. it is safe to say that the combustion chemistry is well understood [51–54]. higher blends require engine modifications and. Moreover. in some cases. Figure 1. The oxidation of these fuels proceeds along the well-established mechanisms summarized in classical texts [49. manufacturers have introduced so-called ‘flexible-fuel vehicles’ that are able to run on fuels ranging from pure gasoline to E85. 1.

The solid and dashed lines indicate computations from two different groups. 134.5 0 2 2000 100 C2H5OH CO2 Mass fraction 10-1 H2O O2 10-2 H2 10-3 0 5 CO 1600 Temperature (K) 1200 800 400 r/rp 10 15 Figure 1.12 Structure of the Bioenergy Business 0. The vaporization rate is presented in the top figure in terms of the rate coefficient Kb. Detailed modeling of an isolated ethanol droplet combustion under microgravity conditions. along with the mass fraction of water in the liquid phase. J.8 0. 301–314. A. and F. There are two fundamental initial decomposition mechanisms of the alcohol molecules that occur during the combustion process. In a first pathway.6 Kb (mm2 s-1) . Copyright 2003. Reprinted from Combustion and Flame. In the alternative pathway.5 1 Time (s) T 1.4 Vaporization rate and species computations for ethanol droplet combustion based on two different ethanol mechanisms. with permission from Elsevier Ref.4 0. a very convincing agreement between species and droplet evaporation rates for ethanol droplets in microgravity for computations performed with mechanisms proposed by two different research groups. Dryer. L. because inefficiencies there can lead to incomplete oxidation and subsequent release into the atmosphere of particularly dangerous pollutants. and an oxygenated radical is formed that ultimately leads to the formation of an aldehyde. Kazakov. This points to the need for a highly efficient oxidation process during power generation.2 0 0 0. A crucial transport phenomenon that couples with combustion chemistry is the strong capability – especially of Liquid water mass fraction 0. Conley. the hydroxy group is displaced from the alcohol molecule and an alkyl radical forms that ultimately can lead to the formation of an olefin. such as formaldehyde and acetaldehyde. the alcohol molecule is attacked by radicals at a location different than the one of the –OH bond. for example. The bottom figure shows the distribution of several species mass fraction and temperature as a function of the nondimensional distance from the droplet center.4 0. [54]) shows.

To this end. The isomers of butanol are shown in Figure 1. 1. the interest in butanol use as a transportation fuel is sustained [55]. compared to gasoline.4. This phenomenon is of course included in the modeling of practical combustion calculations. Alternatively. the two general pathways described above lead to the formation of acetaldehyde and ethylene as major intermediate species. butanol produced from the ABE fermentation processes) contains only three of the isomers and does not include tert-butanol. The kinetic path that leads to acetaldehyde formation is as follows: C2 H5 OH þ X ! CH3 CHOH þ XH CH3 CHOH þ M ! CH3 CHO þ M þ H CH3 CHOH þ O2 ! CH3 CHO þ HO2 where X and M denote combustion intermediates that can act as collision partners.5. As a result..). The combustion kinetics of heavier alcohols has. An interesting characteristic of ethanol is that. as ethanol is an oxygenated fuel and thus one would expect a soot-less combustion. Focusing on the particular case of ethanol. most currently available biodiesels consist of fatty acid methyl esters (FAME) with alkyl chains that are typically 16–20 carbon molecules long. perhaps intuitively.Characteristics of Biofuels and Renewable Fuel Standards 13 light alcohols – to absorb water vapor. This characteristic of alcohol fuels to ‘re-absorb’ in the liquid phase a product that was released during combustion is unique. it is expected that mechanisms as reliable as those already described for ethanol will be rapidly established for other alcohols if. The oxidation mechanisms of these chemicals can differ substantially.2 Biodiesel: Esters As explained in detail in Section 1. which is a product of the biodegradation of methyl tert-butyl ether (MTBE). as intermediates of the oxidation of each individual component can affect the oxidation of the others.2. Nevertheless. However. Detailed kinetic modeling of such molecules is a task beyond current computational .1. the determination of detailed kinetics for blends as opposed to pure chemicals constitutes a crucial step for the detailed study of biofuel chemistry.e. ethanol flames produce significantly increased amounts of soot at slightly elevated pressures (starting from as low as 2 atm. a complicating issue is the existence of alcohol isomers. on the other hand. the ethanol molecule can be attacked by atomic hydrogen and yield an ethyl radical that ultimately produces ethylene: C2 H5 OH þ H ! C2 H5 þ H2 O C2 H5 ! C2 H4 þ H The relative importance of these two routes apparently depends on the overall stoichiometry and. the chemistry of a complex fuel mixture is not simply the sum of the chemistries of each of the constituents. [56] will be instrumental in guiding the modeling of the oxidation process of this alcohol. received significantly less attention. It is noted that bio-butanol (i. Notably. the ethylene pathway is preferred over the acetaldehyde pathway in mixtures that are richer in fuel. the recent detailed measurements of an impressive variety of intermediate species for butanol oxygen flames by Yang et al. This is rather counterintuitive. for example.

a molecule that has a carbon chain almost four times shorter than the esters typically encountered in biodiesel. Remarkably. a recent study by Huynh and Violi [61] is very interesting because it points to the possibility of ab initio calculations of biofuel combustion chemistry. the model consists of a little less than 300 species and 1500 chemical reactions. .” [57]. This generates the question of whether the modeling of methyl butanoate kinetics that has since followed is to be envisioned as targeting the qualitative characteristics of long-chain methylester combustion. [60] indicated a negative temperature coefficient behavior (essentially the chemical process that determines ignition delay). The implied undertaking is clearly nontrivial: the most comprehensive mechanisms that have been described to date pertain to methyl butanoate. moreover. then perhaps the results of methyl butanoate studies are more important in terms of development of methodologies rather than in terms of. which the advanced modeling of butanoate in Refs [58. or whether it is rather a prerequisite for the development of the tools that will ultimately address this issue in the future. If such an important characteristic of combustion chemistry cannot be effectively captured by the kinetics of the surrogate. The establishment of this methodology as a foundation of multiscale computations that would ultimately lead to the description of much-needed mechanisms to be used as input for engine-combustion codes such as KIVA would be a major contribution. In view of this. The justification provided for this approach in the first study reporting a detailed mechanism for the oxidation of this chemical was that “. starting from no less than quantum chemistry of the fuel molecule. emission or ignition delay modeling for biodiesel. the motored engine experiments with the long-chain ester (decanoate) reported by Szybist et al.14 Structure of the Bioenergy Business OH OH 1-butanol CH3CH2CH2CH2OH iso-butanol CH3CHCH2OH CH3 OH OH CH3 2-butanol CH3CH2CHOH CH3 Tert-butanol CH3COH CH3 Figure 1.although methyl butanoate does not have the high molecular weight of a biodiesel fuel. 59] did not show. it has the essential chemical structural features namely the RC(¼O)OCH3 structure. for example. it has only been tested for pressures that are significantly lower than those under which diesel combustion occurs in a typical engine! An alternative approach to ab initio calculations can be the one recently proposed by . Recent results [58–60] have indicated that methyl butanoate is of limited use for the quantitative simulation of biofuel combustion. methyl-butanoate [n-C3H7C(¼O)OCH3] has been used as a surrogate for the purposes of detailed modeling. . In this context.5 The molecular structures of butanol isomers capabilities. Specifically.

1.e. and a detailed report on the issue is provided in Ref. although oleate exhibits a very intense light-induced acceleration of oxidation. to a very significant extent. These are unstable substances that can form a variety of secondary products of either smaller (acids. This observation suggests that it could be possible to delay.. which combines a methyl butanoate mechanism with a skeletal kinetic mechanism for hydrocarbon combustion. It is only reasonable to expect that heavier and potentially more toxic oxygenates may also appear during the combustion of heavier fuels. with linolenate being more vulnerable to auto-oxidation than linoleate and oleate. [62]. although it has been pointed out that even small amounts of unsaturated components also can cause serious problems. The underlying fundamental processes involved have been reviewed in detail by Knothe [63]. and is thus able to show negative temperature coefficient behavior. aldehydes) or larger molecular weights (dimers). and on the other hand the parallel involvement . the involvement of multiple chemical reaction mechanisms. and acetaldehyde. The development of appropriate quantitative analysis methods is difficult given. such an approach would be computationally less intensive. These components are unstable with regard to oxidation for long-term storage. methanol. At the heart of the matter lie the unsaturated components (i. Although oxidation is a widely recognized problem. The primary products of this type of oxidation are allylic (i. but other antioxidants have also been proposed that are specific for biodiesel storage. the conclusions based on methyl butanoate studies that can be reasonably expected to hold true even for heavier esters is the presence of several light oxygenates in the combustion intermediates. Macroscopically.2 Oxidative Stability An additional issue that relates to biodiesel oxidation is that of its oxidative stability. These oxygenates may constitute novel pollutants when one compares these to the ones emitted from the combustion of nonoxygenated fuels.Characteristics of Biofuels and Renewable Fuel Standards 15 Brakora et al. and they undergo both auto-oxidation and photo-oxidation. to this date there are no widely accepted quantitative measurement methods to control the quality of biodiesels with regards to the extent of oxidation that they have undergone. (ii) an elevated temperature.e. thus leading to photo-oxidation. on the one hand. There is evidence that all these fuels generate several pollutants. oxidation with appropriate additives that could compensate for the lack of naturally occurring antioxidants. but the establishment of ab initio calculations would offer the exciting possibility of chemically tailoring the fuel for optimum emission characteristics. Naturally occurring antioxidants are substances that have vitamin E activity.. With these methodological issues in mind. by (iv) exposure to light. Proneness to both auto. (iii) exposure to air and.4.. these chemical processes manifest themselves through an increase in viscosity and the corrosion of engine components. A major conclusion is that oxidation can be catalyzed or inhibited by minor components present in fuel mixtures. Clearly. which can facilitate oxidation by as much as 30 000 times.e. [64]. including formaldehyde.and photo-oxidation varies with biodiesel FAME types. but not avoid. organics of the form R–O–O–H). which are substantially present in soybean and canola/rapeseed oil. the components that contain double bonds). The oxidation is facilitated by the presence of: (i) metal impurities. containing the group CH2¼CHCH2-) hyperperoxides (i.

what parameter is kept constant when comparing various fuels. However. the total energy content of the mixture.1) help to reduce carbon monoxide emission levels. Furthermore. etc. such as O. the underlying issue is that. as a result. Recent chemical kinetic modeling with reactions describing soot formation have provided a more detailed description of soot formation processes from oxygenated fuels [65–67]. while the second proceeds via the presence of high concentrations of radicals. the NOx emissions decrease when the biofuel content increases (up to a local minimum at 50 % biodiesel per volume). As a result. appropriate engine and fuel designs must be employed in order to minimize biodiesel emissions. the total mass of fuel injected. oxygenated diesel fuels have been found to effectively reduce soot emission as compared to conventional diesel combustion. diesel and biodiesel do not have the same energy content and physical properties.3 Emissions The presence of oxygen in biofuels is beneficial as it reduces harmful exhaust emissions to a significant degree.5.e. More than any other oxygenated fuels. when an early injection scheme is used. there is a monotonic increase of NOx emissions with increasing biodiesel content. The situation is not worrisome. this particular problem has perhaps not yet received the attention it deserves. For example.. However. Another issue is that of carcinogenic oxygenated emissions in the form of light ketones and aldehydes. the high oxygen content of methanol and ethanol (see Table 1. Like biodiesel. this hypothesis is invalidated by experimentation. thereby limiting the availability of carbon for soot precursor formation. by 25–30 % according to the US Environment Protection Agency. Another important issue relates to the production of NOx upon biodiesel combustion. the injection strategy can also be important [68–71]. No blanket statement can be issued for these phenomena. these radicals promote carbon oxidation to CO and CO2. The initial intuitive expectation is that oxygenated esters are characterized by increased NOx emissions when compared to nonoxygenated. as long as ethanol remains an additive at relatively low levels and in the order of 10 %. Methanol or ethanol gasoline blends also dramatically reduce emissions as compared to conventional fuels. in general. when using the classical scheme of a late single injection. although it may have to be revisited if E-85 or E-100 . A discussion concerning the determination of the oxidative stability of biodiesel fuels is included in Section 1. rather. In turn. Finally. the total engine load. and HCO. The first of these mechanisms proceeds via a natural shift in pyrolysis and decomposition products. numerical modeling has shown that oxygenated diesel fuel reduces the production of soot precursors–and therefore also soot and particulate matter (PM)–through several key mechanisms. All of these factors contribute to a reduction of soot due to the presence of oxygen in biodiesels. petrol-derived diesel. the effect of a longer ignition delay (due to the fact that biofuels have a higher boiling point than petroleum-derived fuels) competes with the effect of a higher oxygen content and.16 Structure of the Bioenergy Business of physical chemical phenomena. Importantly.4. Here. one has to use caution regarding the basis chosen for the comparisons that are performed (i. In particular. OH. which result from the addition of oxygenate compounds.). 1. high radical concentrations (primarily OH) serve to limit aromatic ring growth and soot particle inception.

efficiency. has published specifications for quality that apply up to 25 % anhydrous ethanol content in gasoline. and has been revised a number of times to address issues such as oxidation stability. and China. The American ASTM D6751 standard was first published in 2002. Most countries have tended to develop standards similar to. The ASTM D5798 standard was first published in 1999 to address the quality specification for E85. the Energy Policy Act of 2005 both abolished the gasoline marketers’ obligation to use MTBE and provided no MTBE liability protection. The ASTM D4806 standard specification covers anhydrous denatured fuel ethanol intended to be blended with unleaded or leaded gasoline at 1 to 10 volumetric percentage for use as a SI automotive engine fuel. or that refer to. Utilization of the chemical was boosted in the US by the requirements on oxygenated components for gasoline that were mandated by the Clean Air Act Amendments of 1990. Australia.g.2. Likewise. In this context. durability. Common blends of ethanol with gasoline in the US are E10 (10 % ethanol and 90 % unleaded gasoline) and E85 (85 % ethanol). A summary of the key properties specified in the standards that address the characteristics of ethanol and its production is provided in Table 1. and (ii) parameters related to production and storage. This issue may have to be revisited if heavier alcohols (e. the two primary biofuels that have been commercialized to date. However. butanol) emerge as widely used biofuels and oxygenated additives because of the toxicity of their aqueous solutions. and is used as a basis for standards in a number of other countries such as Canada.5 Biofuel Standards Fuel quality standards are vital in order to ensure engine–fuel compatibility and reliability. In fact. as well as ensuring that their engines are optimized with regards to performance. and meeting emissions regulations. This standard covers a fuel blend. the experience from neat-ethanol vehicles in Brazil cannot be transferred to the US because the emissions regulations there are less stringent [72]. Ethanol is currently used as an oxygenate additive to gasoline. The European Union Standard EN 14214 was approved in 2003. A European Standard (EN 15376) for undenatured ethanol as a blending component for gasoline up to 5 % was finalized in 2008.Characteristics of Biofuels and Renewable Fuel Standards 17 technologies are to be generalized. The EN14214 and ASTM D6751 specifications have similar requirements in order to regulate . This standard has been in place since 1999. Standards have been developed in a number of countries for ethanol and biodiesel. a leading ethanol producer and user. the American ASTM D6751 and the European Union EN 14214 standards.. Standards for biodiesel are well established in many countries. These standards cover two types of characteristics: (i) properties directly affected by the fatty acid (FA) profile of the biodiesel. The latter’s use as an octane enhancer was initiated during the late 1970s with the purpose of gradually substituting lead. although its performance is inferior to MTBE with regards to combustion chemistry. Brazil. MTBE was quickly shown to be a carcinogenic groundwater pollutant. Engine manufacturers depend on fuels meeting these standards in order to be able to address warranty issues pertaining to the fuel. nominally 75 to 85 volumetric percentage denatured fuel ethanol and 25 to 15 additional volumetric percentage hydrocarbons for use in ground vehicles with automotive SI engines. 1. An additional important issue that relates to ethanol emissions concerns ethanol substituting MTBE.

Maximum content specified where denaturant may affect engine. a sample of the fuel under consideration is exposed to a hot air stream at a temperature of 110  C for a period of several hours (minimum 6 h). Sulfates have been associated with fuel injection problems. fuel injectors. A proposal for a US standard that would operate on a similar principle (ASTM D6751) has recently been rejected. As mentioned above.3. acetic acid) are very corrosive to a wide range of metals and alloys. and an extensive compendium of experimental data has been .2 Key properties of ethanol in quality standards. The key properties specified in the standards and that address the characteristics of biodiesel and its production are listed in Table 1. This phase separation varies with changes in ethanol content. Low levels of strong acids such as sulfuric-based acids might not always be detected by the acidity test. Gasoline addition of 1 % is commonly used. Chlorides are corrosive to metals in fuel systems (e. it is clear that organic acid content is only one of the consequences of oxidative instability.. the relatively limited oxidative stability of biodiesel has been a major issue for engine manufacturers. Solvent washed gum contributes to deposits on the surface of components such as carburetors. Methanol content Solvent washed gum Sulfate Water content Inorganic chloride content Copper content Acidity pH levels of contaminants and the effect of different source materials on fuel quality. intake manifolds and valves. Low-molecular-weight organic acids (e. In this assay. The volatile compounds generated as a result of the oxidation by the hot air stream contain organic acids that are collected in a beaker of deionized water. The European biofuel standard EN 14214 includes a standardized test of oxidative stability (EN 14112) that is a Rancimat apparatus test. stainless steel). and there is no universal acceptance on whether a Rancimat apparatus test is the proper way to test stability.g. The presence of copper can increase the rate of gum formation because it acts as a catalyst in the low-temperature oxidation of hydrocarbons.. There is considerable industrial activity on the matter. Higher ethanol contents are desirable from a vehicle operability point of view.g. The water content of ethanol for blending with petrol must be limited to reduce the risk of gasoline separating from the ethanol. Methanol corrodes metals and causes elastomers to deteriorate.18 Structure of the Bioenergy Business Table 1. but increasing the purity beyond a certain point can have significant production cost implications with potentially few operational advantages. the conductivity of which is recorded as a function of time. A minimum ethanol content is required to provide proper combustion and to ensure that other components that may have detrimental effects on operability or fuel performance are minimized. and their importance Properties Denaturant content Ethanol content Importance Minimum content to ensure that the denaturant is effective. These specifications are for pure biodiesel (B100) prior to use or blending with diesel fuel. However. and can contribute to the corrosion of some fuel system parts. temperature and the level of aromatic compounds in the gasoline.

In the case of biodiesel. Phosphorus occurs naturally in some source materials. An indicator of free fatty acids which cause fuel system deposits and reduced life for fuel pumps and filters.3 Key properties of biodiesel in quality standards. biodiesel can be produced from any vegetable oil or animal fat. Property affected by both fatty acid profile and production. and their importance Properties Flashpoint Viscosityc Cetane numberb Cloud point and cold filter plugging pointb Oxidative stabilityb a 19 Importance Fire safety.Characteristics of Biofuels and Renewable Fuel Standards Table 1. The quantities that are measured in some of the proposed tests are summarized in Table 1.4. While ethanol is a single-component fuel in contrast to gasoline and diesel fuel. ethanol and biodiesel are blended with petroleum-based fuel mostly in relatively small percentages. Cetane numbers of biodiesels tend to be higher on average than for diesel fuel. ethanol and biodiesel are the primary alternatives to gasoline for SI engines or diesel for compression-ignition engines. These contaminants poison emission control after-treatment systems. This is a major concern for engine manufacturers because biodiesel has a reduced stability compared to diesel. Metal contaminants arise from fuel production. Ability of fuel to flow through fuel injection system at cold temperatures.6 Perspective Biofuel production is a rapidly growing industry in many parts of the world. and may be regarded as next-generation fuels. and comprises a mixture of saturated and unsaturated fatty acid esters that can have a substantial effect on the properties of the fuel. Results from a lack of quality control in fuel production. B100 has a higher flashpoint than diesel. Property affected by fatty acid profile of source material. . existing compression-ignition engines can run on a 100 % blend (B100). although higher percentages of ethanol (typically up to 85 %) can be used in flexible-fuel vehicles. At present. assembled by Lapuerta et al. Contaminants can cause storage tank. An important issue for countries with cold winter weather. and engine fouling as well as filter plugging. fuel system. allows for a higher maximum limit than for diesel. Satisfactory combustion and lower emissions. Because of limited manufacturing capacities. 1. respectively. Ensures that engine deposits and corrosion do not occur. biomassderived hydrocarbon fuels and hydrogen in the longer term are currently under investigation. Satisfactory fuel atomization and combustion. Acid numbera Free and total glycerola Metalsa and phosphorusb a b c Property related to production and storage. However. but engine manufacturers are reluctant to go beyond 2–5 % biodiesel in a blend because of fuel stability and fuel quality issues. and is therefore a safer fuel. Other biofuels such as bio-butanol. residual methanol in the fuel substantially reduces the flashpoint. Measures the resistance of the fuel to deterioration during storage and potential for creating deposits in the engine. [73].

[63]) Recorded property Oxidative stability (110  C) Content of FAME with more than four double bonds (% mass) Linolenic acid content (% mass) Iodine value (g iodine added per 100 g of fuel in order to saturate all double bonds) Kinematic viscosity (mm3 sÀ1) Acid value Method EN 14112 ASTM D6751 3 h minimum — EN 14213 4 h minimum 1 max EN 14214 6 h minimum 1 max EN 14103 EN 14111 — — — 130 max 12 max 120 max D445. It can be said that the major advances in electronic technologies have created major opportunities for engine manufacturers to be able to control more precisely the operation of their engines.5 including cetane number.5–5. oxidative stability and cold weather properties. thereby posing the possibility of having an engine that would run on either ethanol or biodiesel. there is substantial room for improvement in the flexiblefuel vehicle concept.0 0. Linked with these advances is the potential to develop sensors that can monitor fuel characteristics and allow the engine to respond to the combustion of different fuels by altering the engine settings so as to achieve optimal engine performance on whichever fuel is chosen by the consumer. In the transition from burning petroleum-based fuels in engines to accommodating the combustion of biofuels in the same engine.20 Structure of the Bioenergy Business Table 1. manufacturers face a dilemma: (i) to invest resources into the development of new engines that have the flexibility of running on either biofuels or petroleum-based fuels or a blend of the two. The development of such technologies can be expected to be costly to begin with. which therefore could imply sufficient flexibility in fuel usage that would span both gasoline and diesel.5 3. ISO 3104/5 D664. such as the oxidation .5 3. Quality standards exist for both ethanol and biodiesel that specify fuel characteristics within limits established through numerous measurements and fuel evaluations. Even the application of HCCI to biofuels has shown some interesting results. 74–101].0 0.5–5. or (ii) to continue the development of technologies such as homogeneous charge compression ignition (HCCI) that may yield a major leap forward in engine emissions reduction and efficiency. 42. it is important to characterize the properties of these biofuels.0 0. HCCI technology represents to some degree the convergence of SI and compression-ignition engine technologies. where the unique properties of biofuels may be leveraged to achieve reduced emissions and more efficient combustion [41. 71.4 Standardized tests of biodiesel oxidative stability (adapted from Ref. In order to ensure compatibility with existing engine technologies. until sufficient market penetration is achieved. EN 14104 1. Although existing flexible-fuel vehicles operate to some extent in this way. Some tests within these standards are still subject to debate and modification.9–6.

References 1. carbon monoxide. with regards to the blending processes with biofuels and petroleum-based fuels.Characteristics of Biofuels and Renewable Fuel Standards 21 stability test for biodiesel. there is growing concern that ethanol combustion generates levels of aldehydes that can be expected to impact air pollution. and is also a potent eye irritant and phytotoxin [104]. Applied Catalysis A: General. Other synthetic antioxidants such as tert-butylhydroquinone have been identified as being very effective [46]. However.. and unburned fuel. These standards address the production. that may have a thitherto unseen consequence on public health and the urban environment. so as to provide a reproducible quality fuel for the consumer. (2007) Processing biomass in conventional oil refineries: production of high quality diesel by hydrotreating vegetable oils in heavy vacuum oil mixtures. Whilst the replacement of MTBE by ethanol was seen as a natural and effective step forward. O’Connor. distribution. The same process applies to cold-weather fuel property improvers. 329. particularly in the case of presently unregulated combustion products. A study of air quality effects of using 10 % ethanol in gasoline in the US state of New Mexico [104] showed increased levels of peroxyacetyl nitrate (PAN) and aldehydes in winter. the biofuel industries in the US and in Europe have made considerable progress towards more effective fuel quality regulation via renewable fuel standards. and even within different batches of biofuels. 103]. biofuels generally have a positive effect in reducing harmful emissions such as particulates or soot. A. Of greater concern from a health standpoint are the emissions of unregulated carcinogenic compounds. 120–129. if this blend were to meet the published fuel quality standards. However.. . G. some uncertainty remains as to whether NOx emissions are increased with biodiesel. Especially in the case of biodiesel. thus. PAN has a major effect on ozone formation. mixing a palm oil-based biodiesel with a soybean-based biodiesel could alter the resultant properties of the fuel. It can be concluded from these studies that further research is needed to establish the long-term effects of the increased consumption of biofuels on atmospheric pollution. In the case of oxidative stability. it would be regarded as acceptable. The oxygen typically bound within the biofuel molecules contributes to cleaner combustion. even in relatively small quantities. strategies for reducing NOx emissions are well established. although cost is a factor that will need very much to be investigated in the selection of the most suitable antioxidants. Yet. In recent years. Huber. For example. even the natural antioxidants in vegetable oils (such as the tocopherols) can have a positive effect [102. the regulation of quality is particularly important for ensuring that a relatively consistent fuel product is marketed. MTBE was phased out because of its toxicity in ground water even in small quantities. P. A substantial amount of research effort has been expended into the development of additives that can address the issues of oxidative stability and cold-weather performance. biological sources for oil to be converted into biodiesel can vary considerably even within the same plant species. such as its oxidative stability and its response to cold weather. Questions arise however. From an emissions standpoint. and should be able to overcome any issues with increased NOx output. and it appears that the engine technology and operational characteristics as a result of injecting and combusting biodiesel play a role in NOx production. For example. storage and sale of biofuels. and Corma.W.

and Wilson. R. A. Canoira. used frying oil and tallow. I. 21.. M. A. Falcon.. (2007) Synopsis of experimentally determined effects of electrostatic charge on gasoline sprays. de Risi. and Ryan. G. 82.S. and Prakash. Vegetable Oil Fuels. A.. and Cathonnet. Effects of fuel physical properties on diesel engine combustion using diesel and bio-diesel fuels. Progress in Energy and Combustion Science. R.... M. X.. R. California. GKS Prakash.D. 971–975. San Diego. (2007) Experimental investigation of the possibility of automotive gasoline spray manipulation through electrostatic field. and Hustrulid. Alternative Fuels. Pischinger.L. T. Anderson. in Beyond Oil and Gas: the Methanol Economy. 3.A. pp. 14 (2).. 17. ASAE Publication 4-82. 529–538. and Cuppett. Matheaus. Chang.R..K. C. and McCormick. J. A. Anderson. Reaney.T. Demirbas. 11. (2007) computational analysis of biodiesel combustion in a low-temperature combustion engine using well-defined fuel properties. Fuel. and Freedman. 53–62.. A. 116 (3).W. Bioresource Technology. D. B. (2000).. G. Atomization and Sprays. A.. P.K. Morin. either straight or in blends. Carlucci. Dagaut. 7438–7443. Ali. and Kyritsis. A.. Goeppert. Personal communication. Bagby.S. (2009) Experimental investigation of electrostatic effects on ethanol and ethanol-diesel blend sprays in atmospheric ambiance. 5. and Park. Kang. 22. III (2003) Cetane numbers of branched and straight-chain fatty esters determined in an ignition quality tester. de Risi. G€kalp.J. A.. D-R. 7. Chauveau. 1557–1564. 14..K. Energy Conversion and Management. American Society of Agricultural Engineers. D. Combustion Science and Technology.. V. (1987) Preparation and Properties of diesel fuels from vegetable oils. N. 10. (2008) Removal of free fatty acid in waste frying oil by esterification with methanol on zeolite catalysts. 48 (11).. C. T. D. Energy Conversion and Management. M.. and Hertz.. S. 35–49. F. G. ( G. eds). 91–101. de Risi. B-G. Chung..L. 80 (1). E. and Navarro.N.F.D. L. (1997) Agriculture and Agri-Food Canada.M. Germany. 16. Fuel. Knothe. S.W. Elegant. 14–34. Bioresource Technology. pp.C. (2009) Progress and recent trends in biodiesel fuels.K.Y. 9. 72 (12). 42 (5). (2008). (2006) The hydrogen economy and its limitations. Saskatoon. and Kyritsis. (1998) Combustion of fat and vegetable oil derived fuels in diesel engines. Y. 12. 50. (1995) Fuel properties of tallow and soybean oil esters. Thesis. C. Improving the cold flow properties of biodiesel. and Jones.. Cambridge Society of Automotive Engineers. A. A. (1995).L. 198–208. Computational modelling of NOx emissions from biodiesel combustion based on accurate fuel properties. T. Alcantara.. C. Reaney. Q. 45. Recep.. Lang... C. 99. 125–164. Biomass and Bioenergy.. A. Y. St Joseph. Hanna. and Kyritsis. E. M.. 15. 434–443. 2762–2768.. (1982) Methylesters of plant oils as diesels fuels. Carlucci. .C. 515–527. (2005).P.... and Huseyin. Energy Conversion and Management. Franco. K-H. Siekmann.C. Bakhshi. 20. 18.. International Journal of Vehicle Design. 50. International Journal of Vehicle Design. D. 499–529. USA. 8. (1998) Carbon cycle for rapeseed oil biodiesel fuels.. P. A. Schwab. G. 176 (4). Journal of the American Oil Chemists Society. Olah. Journal of Engines. C. E. 61–79. Fidalgo. University of Illinois at Urbana Champaign. Selim. and Fernandes.. 66 (10). Biomass and Bioenergy. Maxwell.M.S. 6. Ra.P. A. 133–167. M.C.. Ml. A. Olah. Dalai. 24. D. A. Graboski. 17. 19. A. M. T. and Reitz.22 Structure of the Bioenergy Business 2. 18 (6).P. and Lee.. R. E. 1372–1378.K.B. 91st AOCS Annual Meeting. D. Wiley-VCH Verlag.W. Anderson. (2001) The potential of using vegetable oil fuels as fuel for diesel engines.P.C. 23. Goeppert.C..O. (2000) Catalytic production of biodiesel from soy-bean oil. McCrady. J. 13.L. Stringer.. (2007) Electrostatic effects on gasoline direct injection in atmospheric ambiance. SAE Paper 2008-01-1379.. Yuan. M. Peterson. Amores. Saskatchewan. 289–313. 4. (2001) Preparation and characterization of bio-diesels from various bio-oils. Weinheim.. Ph..C. and Kyritsis. Hansen.J. 24 (2). Huang. Carlucci.. J. (2004) Vaporization and o oxidation of liquid fuel droplets at high temperature and high pressure: application to N-alkanes and vegetable oil methyl esters.

2801–2808. J. D. Zeng. 29. and Lee. Biomass and Bioenergy. (2008). 37. and Wang. 2185–2193.H. 16 (6).J.E. G. and their blends. 43. (2005). (2007) Fuel property effects on injection timing.. (2002) Preferential vaporization model for multicomponent droplets and sprays using continuous thermodynamics.F. Proceedings of the 15th Annual Conference on Liquid Atomization and Spray Systems. diesel.L. and Lee. Journal of Engineering for Gas Turbines and Power. 40. C. 77 (2). C. Cheng... L. Wang. Wang.. and Thompson. and Lee.. J. 42.. C. 50 (4).E. Y.F. Y.F.S.-W. and Lee. W. J. Wang. (2003) Continuous thermodynamics finite diffusion model for multicomponent fuel spray evaporation. Journal of the American Oil Chemists Society. Zeng. Combustion and emissions of biodiesel and diesel fuels in direct injection compression ignition engines using multiple injection strategies. and Lee. C. Tat. 1511–1513. Cheng. (2007) Modeling droplet breakup processes under micro-explosion conditions. (2009). P. Journal of the American Oil Chemists Society.G. continuous multicomponent fuel film vaporization model for multidimensional engine modeling. and Lee.. 112 (2). Y. J. and Lee. 163–186. 45. Stringer. 28. C.F. (2003) Speed of sound and isentropic bulk modulus of alkyl monoesters at elevated temperatures and pressures. Y. V. M. W... and oxides of nitrogen emissions from biodiesel-fueled engines. 964–973. 1365–1370. 255–269.L. SAE Paper 2008-01-1388. and Lee. ignition timing. SAE Paper 2005-01-0209. C. 34. (2001) A Micro-Explosion Model for Multicomponent Droplets.L. Lee. (2002) A preferential vaporization model for multicomponent droplets and sprays. 1123–1128.F.C. Tat.L. Wang. Tat. 32. D. D. Comparing the operation of an HSDI engine using multiple injection schemes with soybean biodiesel. D.. 51 (4). Journal of the American Oil Chemists Society. diesel and their blends.F.F. (2008) Finite diffusion wall film evaporation model for engine simulations using continuous thermodynamics.F... Chiu. Proceedings of the Combustion Institute. A.F. C.E. and Hansen. A.E. (2000) The specific gravity of biodiesel and its blends with diesel fuel. Van Gerpen. Journal of Engines. Wang. and Van Gerpen. Proceedings of the 16th Annual Conference on Liquid Atomization and Spray Systems. Proceedings of the 14th Annual Conference on Liquid Atomization and Spray Systems. 485–491.F. Tat. M. . W. C. Cheng. and Van Gerpen. C. 31. C. C. Transactions of the ASABE. Lee.F. 27 (5).F. (2004) Impact of cold flow improvers on soybean biodiesel blend. Proceedings of the 32nd International Symposium on Combustion.F.S.H. 26..H.F. C. Proceedings of the 13th International Engine Combustion Multidimensional Modeling Conference..H. 35. 76 (12). 32. Schumacher. 44. (2000) Multicomponent-fuel film-vaporization model for multidimensional computations. Shrestha. 30. Atomization and Sprays. Y. Van Gerpen. (2009) Finite diffusion wall film evaporation model for engine simulations using continuous thermodynamics. Proceedings of the Central States Section Meeting of the Combustion Institute.. and Lee. (2002) A model for multicomponent spray vaporization in a high pressure and high temperature environment. SAE Paper 2009-01-0719. D. and Lee. Proceedings of the Combustion Institute.Characteristics of Biofuels and Renewable Fuel Standards 23 25. and Lee. 27. C. D. D. 12 (1). Wang. Zeng. Zeng. M. C. Transactions of the ASABE. V. (1999) The kinematic viscosity of biodiesel and its blends with diesel fuel.. Y. Wang. 41. C. 31.L. 80 (12).C. Zeng. 38. and Suppes. 1249–1256. 33. J. Zeng. (2003) A model for droplet and spray vaporization under elevated pressure conditions using continuous thermodynamics. (2002) A model for preferential vaporization of sprays of complex liquid mixtures using continuous thermodynamics. (2003) Modeling of air fuel mixing in a stratified gasoline direct injection engine using multicomponent fuel representation. and Lee. Stringer. M. Journal of Propulsion and Power.L. Wang. D. Cheng. 717–724. J. 36. 115–119. D.F. and Lee.. and Hansen. W. 39. C.H.. 124. (2008) Effectiveness of cold flow additives on various biodiesels. and Van Gerpen.

(2000) Detailed chemical kinetic mechanisms for oxygenated fuels. C.M...S. S.. M. SAE Paper 2008-01-1378.. 57.. and Smith. 58..L. 50.C. Wang.. 63. Norbeck. SAE Paper 2008-01-1379. Reitz. J. Bhale. Westbrook. . CA. Fuel Processing Technology.S. J. J. (1992) Kinetic modeling of ethanol pyrolysis and combustion.. J. B. and Blaschek. A. Ra.. and Lee. Kinetic modelling. 134. Development and validation of a reduced reaction mechanism for biodiesel fueled engine simulations. C.. H.... 823–833. Dagaut. T. T.J.J. McFarlane. Knothe. Curran. Sarathy. 53. J.. Szybist. A.. L. J. and Kohse-H€inghaus.R. F. Li. R. Technical Report NREL/TP-540-39721.A.M.. and Simmie. (2008) Thermal decomposition of methyl butanoate: Ab initio study of a biodiesel fuel surrogate. (2006) Analyzing biodiesel: Standards and other methods. C. 94–101. Journal of the Chemical Society Faraday Transactions. 153. 52. 867–884. and Westbrook. S. (2008). 62. Boettner. 88.C. 729–731. R. Fisher.L... 794–800. P. Law. Tian. A.P.24 Structure of the Bioenergy Business 46.. Proceedings of the Combustion Institute.. Collins. N. and Dryer. 24. J.. (2003) Detailed modeling of an isolated ethanol droplet combustion under microgravity conditions. 47. R. Y. G. J. 319–344. Presentation at Diesel Engine-Efficiency and Emissions Research (DEER) Conference. 305–311. M. 66. Proceedings of the 20th Annual Conference on Liquid Atomization and Spray Systems. Oils and Related Materials. Y. Syed. Patterson.. M.M. 48. McFarlane. Glassman.P.. Stability of Biodiesel and biodiesel blends: Interim Report.W. emissions and emission control. Ra.. Marchese. S.. International Journal of Chemical Kinetics. Z. and Porter. (2009) Improving the low temperature properties of biodiesel fuel. (2008). Thompson. Durbin. Stringer. fuel rich.V.. Yang. K. Alleman.. Waynick. and a synthetic diesel on emissions from light heavy-duty diesel vehicles.. 51. Pitz. 56.L.L. 34 (22). P.J. 1579–1586. R. and Thombre. S.J. (2006) Combustion Physics. W. M. 2–32.V. Kazakov. Alam. Wei.. N. P. T. Journal Chemie Physie et Physico-chimie Biologique. and e Dryer. 89. Conley. and Dryer. Divart. H.A..K. J. P. McCormick. Process Biochemistry. Cheng. B.. J. 2549–2559..D. and Buchholz. 64.. 59. Abacus Press.M. (2007) Biodiesel combustion. (2000) Effects of biodiesel.A. 54. (1991) High-temperature oxidation of ethanol: 2. 17 (11)... (2006). 68. International News on Fats. Effects of fuel physical properties on diesel engine combustion using diesel and bio-diesel fuels.D. F. T.. M. G. Cambridge University Press. Journal of the American Oil Chemists Society.J.. 349–355.R. (2008) Autoignition measurements and a validated kinetic model for the biodiesel surrogate. 34 (3). J. J.D. (2007) Modeling biodiesel spray breakup with well-defined fuel properties.M. 61. May 2007.P. H. Combustion and Flame. (2007) o Identification of combustion intermediates in isomeric.L. National Renewable Energy Laboratory.L. J. Hansen. Song. J. Brakora. 60. Norton. SAE Paper 2002-01-1705 69. (2006) Biodiesel and vegetable oil fuels: then and now. The effect of oxygenates on diesel engine particulate matter.. San Diego. Dibble.S.L.. McCrady.S. methyl butanoate. (2002). (2007) A wide-ranging kinetic modeling study of methyl butanoate combustion.K. biodiesel blends. L. 55. IL. S.. A. C. and Cathonnet. 67. (1987) Combustion. Proceedings of the Combustion Institute.K.. Dagaut. Deshpande. Forbes. 34–39.P.. 679–691. Qureshi. F. Cahicago. August 20-24. Renewable Energy. K. Reitz. Knothe. 65.. Eseji. P. C. 73. Qi. Dunphy. 28. 87.F. 49. and Daw. and Boehman. Journal of Organic Chemistry. Detroit.. F..B. premixed butanol-oxygen flames at low pressure.M. V. 31. Combustion and Flame. McCormick. 42. Environmental Science and Technology. 301–314. Oswald. and Daw. Huynh.. and Violi. Alternative fuels. 148. (1992) An experimental and modeling study of ethanol oxidation kinetics in an atmospheric pressure flow reactor. MI. Combustion and Flame.. 198–209.R. Gail. 83 (10). Y. (2007) Production of acetone-butanol-ethanol (ABE) in a continuous flow bioreactor using degermed corn and Clostridium beijernickii. S. and Simmie.L. A. Curran. R.. P. Dooley.. I. January 2008. A.. S..

Fang. 34. A. 71. (2009) Influence of injection parameters on the transition from PCCI combustion to diffusion combustion mode within a small-bore HSDI diesel engine. V. Combustion and soot visualization of low temperature combustion within an HSDI diesel engine using multiple injection strategy. (2008) Effects of injection angles on combustion processes using multiple injection strategies in an HSDI diesel engine. (2007) Modeling droplet breakup processes in bio-fuel diesel engines under micro-explosion conditions. T.A. Hansen. Physical property measurement of biodiesel fuels for low temperature combustion modeling.L..A. T. O. R. and Lee. T. Cheng. (2008) Effect of biodiesel fuels on diesel a engine emissions. and White. Coverdill. 42.. J. McCrady. 2785–2792. Stringer. Emissions from ethanol. Proceedings of the Combustion Institute.E. C. Stringer. (2009) Fuel effects on combustion processes in an HSDI diesel engine using advanced injection strategies. (2005) High-speed Mie-scattering measurements of diesel sprays under MK combustion mode within a HSDI diesel engine.F. Lee... T. SAE Paper 2005-01-3838. Coverdill.F. SAE Paper No.. Liquid and vapor fuel distributions within a high speed direct injection [HSDI] diesel engine operating in HCCI and conventional combustion modes. R. Coverdill. Fang.. C. and Lee. C.L. Y. R.C.F.F. Pitstick. Ph. R. Study of soot formation of oxygenated diesel fuels using forward illumination light extinction [FILE] technique.. University of Illinois at Urbana-Champaign. 8865–8870. and Lee. Computational analysis of biodiesel combustion in a low-temperature combustion engine using well-defined fuel properties.F. Low-temperature combustion within a small bore high speed direct injection [HSDI] diesel engine.. 10 (3). Wang. (2005). R. V. J. McCrady. Department of Mechanical Science and Engineering. M. Cheng. Lin. C. R..E.. and White. Lin.F.F.D.. Proceedings of the 20th Annual Conference on Liquid Atomization and Spray Systems. W. Lee. Lee. C... Armas.C. 32.E. 77. 72.P.. Coverdill. R. T. Atmospheric Environment. C. 2008-01-1388. Fang. R. SAE Paper 2006-01-1415. C.F.. and Lee. Fang.-C.. and Hansen. and White. Lee. 198–223. Proceedings of the 18th Annual Conference on Liquid Atomization and Spray Systems. C.F. 1133–1143. 74. Hansen. (2005). J. Environmental Science & Technology. W. 76. T. 80. 87. and Lee. Fang. ANL/ES/PP 79436 Argonne National Laboratory.. Lee. 3232–3239.. Coverdill. C.. (2008) Characterization of particle size distribution from diesel engines fueled with palm-biodiesel blends and paraffinic fuel blends.C. T...E.F. T.F. C. and Lee. Fang. Proceedings of the 20th Annual Conference on Liquid Atomization and Spray Systems. and White. 82.F. Fang. Lee. Report Nr. Thesis.. Xu. (2009) Low-temperature combustion within an HSDI diesel engine using multiple injection strategies. (2007).A..L. T.E. (2008).. R.E...F. (2006). 87. A. SAE Paper 2007-01-0617.. R.F. Fang.A. Low temperature combustion within a small-bore high-speed direct-injection optically accessible engine. 73. 88... R. Coverdill. R. (2007). T..F. (2006).C. Fang. J. T. Lee... C. and Lee. Lee.. A. 83.P. SAE Paper 2006-01-0078.-C. T.F. and Rodr ıguez-Fernndez. Foong. International Journal of Automotive Technology. V. 78. 42. 81. Fang. Coverdill. (2008) Reducing NOx from the biodiesel-fuelled engine by low-temperature combustion. R.. 285–295.E. C. R. A.. C. K. Combustion and emissions of biodiesel and diesel fuels in direct injection compression ignition engines using multiple injection strategies. Journal of Engineering for Gas Turbines and Power (accepted for publication). and White. C. Lapuerta. and White.A.T. 75. Fuel.P. Fang.. 85. Fang. M. 86.M..F.L. and White. ASABE Paper 066146.. Progress in Energy and Combustion Science. McCrady. and Lee. and Lee. 84. Proceedings of the Central States Section Meeting of the Combustion Institute. (2006). Hansen. Y.Characteristics of Biofuels and Renewable Fuel Standards 25 70.. (2006) Combustion visualization in an optically accessible HSDI diesel engine using different injection angles.E. (2007) Comparisons between a high speed direct injection engine operating with biodiesel and petroleum based diesel. C.. 89. SAE Paper 2005-01-0919. Stringer. 79. T. .A.. C. Y.A. C..and LPG-fueled vehicles.

C. V.. L. Modeling biodiesel combustion using GTpower.. Journal of the American Oil Chemists Society. Fr€hlich. Cheng. New Mexico.F. ASABE Paper 076095. Proceedings of the 5th Asian Aerosol Conference. Low temperature combustion within an HSDI diesel engine using multiple injection strategies. C. W.. 97.G. C.P. Fang.. 104. 579–585. Lee. (2007). T. 92. and Lee. (2008) The effect of natural and synthetic antioxidants on the oxidation stability of biodiesel. C..L.A. (2008). Fang. J. (2008). Lin..26 Structure of the Bioenergy Business 90. A.. Foong. 94.M. C. D. T. 98. J. McCrady. Kaohsiung. Y. S. Salley.P.-C. 95. Y. (2007) Combustion in an optical diesel engine fueled with diesel and biodiesel fuels using multiple injection strategies. C. and Ruan. and Lee. Environmental Science and Technology. Gaffney. Proceedings of the 20th Annual Conference on Liquid Atomization and Spray Systems. T. T.F.C.W.F. Reyes. ASABE Paper 076096.. and Lee. R. 102.. W. Wang.L.F.. T.. Comparison of performance and combustion characteristics of diesel fuel and vegetable oils in DI diesel engine.C. and Ng. and Lee. J.. Tang.. Computational analysis of the properties of biodiesel blended with diesel fuel. A. and Lee. Proceedings of the 20th Annual Conference on Liquid Atomization and Spray Systems. Fang. ASME Paper ICEF2007-1747. Ruan. (2008). (2007) Characterization of particle size distribution from diesel engines fueled with palm-biodiesel blends and paraffinic-fuel blends. Proceedings of the 21st Annual Conference on Liquid Atomization and Spray Systems. 100. 3053–3061. Cheng..P. R. J. SAE Paper 2008-01-1639. 101. and Popp. T. Taiwan.C.F. C. C. Dixon. C.. Fang. (2007) Modeling biodiesel spray breakup with well-defined fuel properties. and Lee. Fang. 93. and Lee. H. A. A.L. Hansen.. (2008) Fuel effects on combustion processes in an HSDI diesel engine using advanced injection strategies. 91.. Fang. 99. Journal of the American Oil Chemists Society. (2007).. Hansen. 31. Spray and combustion visualization in an optical HSDI diesel engine operated in low-temperature combustion mode with bio-diesel and diesel fuels. . Comparisons of combustion characteristics of biodiesels in a high speed direct injection diesel engine.S.S. Lin. (1997) Potential air quality effects of using ethanol-gasoline fuel blends: a field study in Albuquerque.F. T. Fang. and Schober. 373–382. 84 (6). Stringer. and Lee. N. Proceedings of the Eastern States Section Meeting of the Combustion Institute.C. Marley.F..F.. SAE Paper 2008-01-1390. C. 96. 85 (4). Proceedings of the 32nd International Symposium on Combustion.F. and Lee. S. Martin.F.. A. K.F. C. (2007) The influence of tocopherols on the oxidation stability of o methyl esters. SAE Paper 2008-01-1638. (2007) fuel effects on the spray and combustion processes within an optical HSDI diesel engine. (2008) Spray and combustion visualization in an optical HSDI diesel engine fuelled with biodiesel and diesel using multiple injection strategy. McCrady. McCrady.F.. C.. (2007). and Lee..F. D.F. T. and Lee. C. 103.. Hansen.J.

Whilst ethanol and biodiesel have expanded into the existing markets and infrastructures of gasoline and diesel. 2008). soybeans or canola oil. based on cereals (e. corresponding to 36 million tonnes oil equivalent (MTOE) or 1. In 2007. the global production of biofuels amounted to 62 billion liters (GL: gigaliters) or 16. and a further doubling is expected to occur in the coming decade. or organic waste.6 GL (13. based on vegetable oils such as rapeseed. Remarkably.. in particular bio-butanol and biohydrogen. and 3. palm. Furthermore. great expectations rest on cellulosic biofuels using wood. this trend is forecast to continue. corn) and sugar crops (e. Nasib Qureshi.2 The Global Demand for Biofuels: Technologies.. other renewable fuels have begun to emerge as potentially viable alternatives.1 Introduction Rapid growth has been witnessed in recent years in the production and consumption of biofuels for powering combustion engines for the transportation economic sector. Fuel ethanol accounts for most of the world’s biofuels. Markets and Policies J€rgen Scheffran u 2. 4. grasses. in about 2005 Brazil was overtaken by the US.g.1). Almost half of the ethanol is produced in the United States.1 BG) in 2007.8% of total global transport fuel consumption in energy terms (OECD. and biodiesel. Biomass to Biofuels: Strategies for Global Industries and Hideaki Yukawa Ó 2010 John Wiley & Sons.3% in the European Union. with a production of 49. 38% in Brazil. Hans Blaschek e .7% in China (Table 2.g. a dramatic rise compared to about 20 GL in 2002. The most important biofuels today are ethanol. sugarcane or sugar beet). Ltd Edited by Alain Verts.4 billion gallons (BG) per year. although it remains the largest exporting country. Whilst Brazil was by far the world’s largest producer throughout the 1980s and 1990s.

almost one-third in Germany (EBB. and less than 2% in the European Union (EU) as a whole.4 15.3 486 211. as they help to reduce associated technology commercialization risks.2 4. 2007). 3% in the USA. 2008). these policies significantly restrict the potential of Brazil and other nations to export biofuels (BNDES.1 World ethanol production in 2007. although this phenomenon is largely induced by public support policies rather than by market forces. It has been estimated (OECD. US. the share of biofuels in total transport-fuel demand was about 20% in Brazil.7 13. Based on the assessment . Industry Statistics and F. These policies constitute an important tool to accelerate the pace at which biofuel transportation-related technologies and logistics penetrate the market. In this chapter. of which more than 60% was produced in Europe. widely applied are blending or the use of mandates that require biofuels to represent a minimum share or quantity in the transport fuel market.O.8 9. and one-seventh in the USA (Lane.101. www.70 Global biodiesel production amounted to about 10. EU and Canadian biofuel supply and use in 2006 was about US$ 11 billion per year. In 2007.6 5019. 2008). Biofuels production and use are promoted by an increasing number of countries.7 billion gallons (BG)] in 2007 (OECD. we review the current and projected global demand for biofuels in light of emerging national and international markets and public policies. Ambitious political targets regarding the substitution of fossil fuels by biofuels in the transportation sector have thus been set in a number of countries.28 Structure of the Bioenergy Business Table 2.9 5. that protect the less cost-efficient domestic biofuel industry from lower-cost foreign competitors. including the EU. 2008).6 26. Licht. including direct support or tax concessions. Reproduced with permission from the Renewable Fuels Association. 2008) that support to the US.3 79.2 gigaliters (GL) [equivalent to 2. More indirect measures are in fact trade restrictions. and statistics Country USA Brazil European Union China Canada Thailand Columbia India Central America Australia Turkey Pakistan Peru Argentina Paraguay Total Gallons (Â106) 6498. such as import tariffs. However. These targets aim at attracting public and private investments to stimulate biofuel production and use.8 39.9 52.2 74. and this is projected to rise to US$ 25 billion in the medium term (average for the 2013–2017 period). Besides budgetary support measures.2 570.ethanolrfa.2 7.

The prices of commodities such as transportation fuels exhibit a tendency to revert to their mean values. A UN study (UNEP. which expects rapidly expanding future markets for grain and land resources. 1999).The Global Demand for Biofuels: Technologies. 2006a) has predicted more than 200 000 new jobs in all sectors of the US economy. this would result in 1. Markets and Policies 29 of motivations and potentials of renewable fuels and technologies. support is especially strong from the agricultural community. if realized. although both costs and risks can be reduced by the use of advanced technologies that permit the blending of ethanol and gasoline at the pump. Advanced bioenergy could possibly help to satisfy the growing energy demands of developing countries. thus facilitating the adoption of ethanol. If extrapolated to biodiesel. economic. diesel and kerosene. about 2. The transportation sector is almost completely dependent on energy from fossil fuels such as gasoline. 2.and truck-dependent system would increase the cost and the risk of accidents. Currently. this would represent an increase of the US GDP by $200 billion between 2005 and 2012. An economic study of existing ethanol plants (Iowa State University. as well as to international trade between key importers and exporters. Thus. EU. The role of biofuels in the remediation of greenhouse gases also is included. As a result. and a resulting increase in farmers’ income by $43 billion. as well as potential environmental impacts that will affect future policy decisions. The projected investments of US$ 630 billion by 2030 would translate into at least 20 million additional jobs in the renewable energy sector. Switching from a pipeline transportation system to a rail. the growth in oil prices and a dependence on energy imports from the Middle East have led in recent years to an increase in the demand for alternative energy sources (as was the case following the ‘oil shocks’ of the 1970s). and environmental benefits of biofuels. Home-grown domestic energy sources offer development perspectives to structurally weak rural areas. particular consideration will be given to Brazil. Japan and US policies. and lead to beneficial structural changes in land-use and agricultural practices (Rosillo-Calle and Walter. building on the existing infrastructure of gas stations and automobile technology.2 Motivation and Potential of Renewable Fuels A foundation of the biofuel market is the high level of public support that is usually justified by the expected energy.16 jobs created per million liters of annual production.4 billion people depend on the traditional energy . yet despite this intrinsic cyclicality. Particularly dependent are the developing countries whose oil supplies rely largely on imports. Additional costs are incurred by retrofitting distribution centers to ethanol blending into gasoline (more than $1 billion have been reported at the end of the 1990s) (AMI. the use of fuels based on plant biomass would offer a significant opportunity to diversify the energy sources in the transportation sector. 2006). The Renewable Fuel Association (RFA. 2008) on the potential of ‘green’ jobs found that renewable energy generates more jobs than employment in fossil fuels. 2006a). Estimates suggest that large-scale biofuels production in the US by 2012 would reduce crude oil imports by two billion barrels per year (RFA. which makes that sector of the economy especially vulnerable to disturbances in petroleum price and supply. 2006) estimates that a 50 million-gallon ethanol plant with 75% local ownership would create 220 new jobs. creating new income and job opportunities to the agricultural sector.

municipalities and industry plays a significant . On the other hand. with high commodity prices at the start of the year. The growing demands and subsidies for ethanol and other biofuels have broadened the economic basis for biofuels. Gallagher. dung and wood. Nevertheless. biofuels produced from wheat. Until recently. The coordinates have dramatically shifted during 2008. GHG savings vary significantly across biofuels.30 Structure of the Bioenergy Business uses of biomass. but low commodity prices at the year-end. unhealthy and nonsustainable (Ezzati and Kammen. 2008). Among the reactive measures that were taken at the time by oil-importing countries. With current policy support. These and other factors determine the threshold of profitability of ethanol (FAO. Searchinger et al. the cost-effective use of organic waste material from agriculture. over its whole bioindustrial cycle. In addition. For example. as their global-scale implementation would help accomplish the targets for greenhouse gas (GHG) emission reductions. making biofuels based on current technologies a rather expensive path to energy security and mitigation of climate change (around US$ 1000 per tonne of CO2-equivalents saved) (OECD. in both developing and developed countries. 2008).. electricity generation and transportation fuels. such as the burning of straw. the competitiveness of ethanol will improve with increasing oil prices. and in turn facilitate sustainable development in these regions (Hazell and von Braun. 2007).. the reduction of GHG emissions and use of fossil fuels amount to about 1% of the total. lighting. there is a significant potential for the production of energy crops and the development of innovative technologies to efficiently convert biomass into energy. Sylvester-Bradley. sugar beet or vegetable oils rarely provide GHG emission savings of more than 30–60%. 2008). The global efforts to identify alternatives to fossil fuels originated from the OPEC oil embargos and subsequent price shocks of the 1970s. These differences can be ascribed in part to specific attributes such as sugar content. Despite these programs and increased research efforts. Due to the high productivity of energy crops in tropical and subtropical regions. while ethanol from corn (maize) generally allows for savings of less than 30%. 2006). For example. as set in the Kyoto Protocol and follow-on agreements (Worldwatch Institute. There is an on-going debate as to whether the carbon balance could be even negative if significant amounts of carbon were to be released during land clearing (Fargione et al. 2008. 2001). provided that the costs of input factors (such as corn and fertilizers) do not compensate for these gains. the United States and Brazil implemented a series of incentive programs to encourage the production of transportation fuels made from organic matter instead of petroleum (INFORMA. All of these uses are obviously often inefficient. 2008). including heating. water pumping and other basic needs. locally produced advanced bioenergy (such as ethanol from sugarcane or biodiesel from palm oil) could potentially provide income and employment in rural areas. and in part to fossil fuel inputs (OECD. 2008. for cooking. ethanol produced from sugarcane may reduce GHG emissions by 80% or more relative to emissions from fossil fuels. Concerns about global warming spur the search for low-carbon energy alternatives to fossil fuels. such that market growth became heavily dependent on government policies. 2006). Biofuels are highlighted as being carbon neutral because the carbohydrates used to manufacture these fuels originate from atmospheric carbon fixed by photosynthesis. ethanol has been more expensive than gasoline. 2008. biofuel production grew only slowly through the 1980s and then stagnated during the 1990s as petroleum prices reverted to low levels. Nevertheless.

however. bioenergy systems will have to become fully competitive with fossil energy and avoid some of the current distortions.The Global Demand for Biofuels: Technologies. Adapted from Kaltschmitt et al. in order to promote the US biofuel industry. Africa.3 Renewable Fuels in the Transportation Sector Most studies on energy use in the transportation sector emphasize the growing importance of automobiles in individual transportation. Concerns about the impacts of growing bioenergy on land that is used for food production and the environment require that bioenergy production and consumption is established in a sustainable manner that minimizes these impacts. 2004).2 and references cited therein). thus creates strong incentives for international trade (see below). In addition. The increasing demand for biofuels in North America. Russia and North America.2. such as subsidies for domestic and import barriers on foreign biofuels. The largest potential area for energy crops exists in Africa (124 million ha). and in Asia it is stalk plants. A regional breakdown of the energy potential of biomass is provided in Table 2. whilst in Africa only about 40% is used and in Latin America only about 10% (see Table 2. if the controversy regarding the adverse implications of biofuels continues and if the pressure on fossil fuel prices relaxes. which highlights that the potential is quite evenly distributed. Latin America. In Europe. . as was observed after the oil shocks of the 1970s and again following the economic recession in fall 2008. and this is expected to lead to a large expansion of sustainable energy and energy-saving technologies over the next decade. wood provides the largest share. Markets and Policies Table 2. the production capacity of the land is already overextended. Recent years have brought a dramatic shift in policy support in many parts of the world. Asia. whereas in Africa and Latin America energy crops represent the greatest potential. and the potential supply from tropical and subtropical countries. followed by Latin America (108 million ha) and North America (36 million ha). (2003) Energy potential (PJ aÀ1) Wood Stalk plants Energy plants Dung Total Current use Energy plants (Mio. and the continued dependence on fossil fuels. with around 20 000 Petajoule (1015 J) in each of the five regions: North America.ha) Europe Former USSR 5400 700 3600 300 10000 500 32 Asia Africa Middle East 400 200 — 100 700 — 0 North America 12800 2200 4100 800 19900 3100 36 Latin America 5900 1700 12100 1800 21500 2600 108 31 Total 4000 1600 2600 700 8900 2000 22 7700 9900 1100 2700 21400 23200 10 5400 900 13900 1200 21400 8300 124 41600 17200 37400 7600 103800 39700 332 role in triggering the transition towards more extensive bioenergy uses (Scheffran et al.2 Global bioenergy potential in petajoules per annum (PJ aÀ1). Europe and Japan. and Europe/Former USSR. however.. and beyond. In Asia. This support may fade. 2.

the growth of the biofuel industry has faded in 2008. Under favored conditions. estimated as 6. Given high oil prices and government subsidies. significant uncertainties remain about future projections of the biofuels market. respectively (EIA. by 28% in the USA and by 26% in the EU. which serve as a raw material for biofuel production. About two-thirds of these emissions were associated with passenger transport.3 gigatons of carbon dioxide (GtCO2) in the whole fuel cycle (from well to wheels) (IEA.7 to 85. it would be difficult to finance a green field ethanol-manufacturing project.. have the advantage that their large-scale implementation can be built on the fleet and infrastructure of existing vehicles. hydrogen. including gas stations. 2007). However.4%. Different alternative energy sources and technologies have been considered for transportation. At the same time. 2006). and in particular whether the ethanol market is able to absorb the new supplies without a significant downward price pressure (Gallagher et al. 2008a). car manufacturers. experienced a similar price development as petroleum in 2008. Despite declining processing costs to produce biofuels.32 Structure of the Bioenergy Business From 2000 to 2006.9% and 6. GHG emissions from the transportation sector increased between 1990 and 2004. Unfortunately. 2006a).2%. and up to 75% if the fuel efficiency standards of automobiles were doubled (Worldwatch Institute. and their various blends with fossil fuels. the profit margins for ethanol plants have been shrinking. from 76. Given this price volatility. including petroleum companies owning their own fuel distribution networks. the US consumption by 5%. 2001).1%. fuel distributors. during the next 25 years biofuels could deliver 37% of the fuel needs in the US. due partly to concerns regarding unsustainable practices in biofuel production. In order to establish an economic environment that is suitable to promote the growth of the market for alternative fuel vehicles. the new biofuel industry would require coordinated investments by farmers. but also on incentives to adopt the new technology throughout the transportation value chain.eia. natural gas. In order to overcome the initial costs and barriers. 2006). the production of biofuels is a growth sector of the energy system. Between 2001 and 2005. Under these circumstances. such as biofuels. the global biofuel production doubled and then more than tripled until 2007.9 million barrels per day (EIA. and electricity (either from the grid or from fuel cells). In 2004. gasoline and diesel constituted about 20% of the world’s energy storage and distribution). while consumption in Germany and Japan in the same period declined by 3. or the alternative fuel vehicles (Romm. due partly to the soaring prices of agricultural . 2006c). and the remainder with freight transport (IPCC. Conventional biofuels such as ethanol or biodiesel. from almost US$ 150 per barrel to below US$ 50 per barrel (www. and others to achieve a critical size. transportation. reaching 170 million liters per day. and partly to the economic crisis which culminated in fall 2008 with a significant drop in petroleum prices. 2006b). 2008a). global petroleum consumption increased by 10. future success relies not only on improving energy efficiency and reducing production costs in the conversion of biomass into biofuels (Hamelinck and Faaij. China’s consumption alone increased by 50. it is essential to resolve the ‘chicken and egg’ problem of what comes first: the overall alternative energy infrastructure (including production.doe. food crops such as corn and soybeans. The GHG emissions of the transportation sector amounted to 28% of total emissions in 2000. and 53% of the oil products were consumed in the transportation sector (IEA. Notably.

distribution to gasoline stations accounted for e1. According to Hamelinck and Faaji (2006).34. (iv) the development of new coproducts. 2004). have been e25 per gigajoule (GJ. the short-term production costs for bioethanol on the basis of corn. oil plants grown in temperate zones have greater water and fertilizer requirements than plants grown in tropical zones. 2007). Thus. While sunflowers produce about 800 kg oil per hectare. 2004. and for biodiesel based on rapeseed. the US Congress has imposed 15 billion gallons per year as the upper limit for corn ethanol production. competition for land use with the food production sector will also limit the growth in conventional biofuels production based on food crops. the yields are 1590 kg haÀ1 for Jatropha and 4000 kg haÀ1 for palm oil (Mathews. Zibechi. between US$ 0. compared to corn. hydrogen or electric engines) that will require a new infrastructure and an increase in the international trade in biofuels. and (v) improving the refinery efficiency and cost-effectiveness (Walter et al. technological innovations. In particular. and tax exemptions. rapeseed and sugar beet in the temperate zones. the biofuel industry is subject to an important political risk that derives from uncertainties regarding the support of succeeding governments for biofuel mandates. Higher Heating Value) in Europe and North America. Zuurbier and van de Vooren (2008) reported the annual ethanol yield of sugarcane in Brazil as 180 GJ per hectare.4 per GJ (see previous reference). The competitiveness of ethanol production will depend on large improvements along the production chain. for each 10% increment in bioethanol consumption. an oil price of e100 per barrel corresponds to e16. For example. for instance. the energy crops of sugarcane. while the production costs for Brazilian bioethanol based on sugarcane remained the lowest in the world. Brazil could produce an equivalent amount of fuel by using only an additional 1. Moreover. including: (i) a reduction in energy consumption. among oleaginous plants used for biodiesel production. a more diversified fuel mix is likely in the transportation sector (Gielen and Unander. the energy balance of corn ethanol is less favorable (1. such as a reduction of oil dependence. mitigation of climate damage. at only e11 per GJ. Notably.. 1995) than that of ethanol produced from sugarcane (8. Markets and Policies 33 commodities and the increasing feedstock costs (FAO.40 are required to replace one liter of fossil fuel by biofuels. sorghum and jatropha have advantages. 2005). This does not include additional cost for taxes and distribution. and job creation effects. It has been calculated that in the US.66 and 1. .g. Under tropical climatic conditions. (ii) diversification of the energy supply. 2007).4 per GJ. further cost reductions will be needed for biofuels to compete effectively with gasoline and diesel without subsidy (OECD. 15% of the agricultural area of the country will be needed whereas.The Global Demand for Biofuels: Technologies. Moreover.. 2007). with modernized spark-ignition diesel engines and hybrid vehicles most likely being replaced by more advanced technologies and energy sources (e. 2007). in contrast. Costs and subsidies for biofuels are partly compensated by the expected economic benefits. Similarly. according to Macedo et al. For comparison. In the future. tropical plants achieve higher yields.3–10.. in the US.5% of its land area (Wisner and Baumel. 2008). 2008). according to Shapouri. Since ethanol plants could consume half or more of the US domestic corn supplies to achieve biofuel mandates. (iii) the inclusion of lowcarbon sources. approximately threefold that of corn or wheat. subsidies. in the EU these costs are even higher (Doornbush and Steenbik.

36 BG). although trade today is growing. to match the predicted ethanol demand for 2030. plan to increase ethanol production by 5 GL or 1. but also significant ethanol price reductions. etc. not only are significant technological improvements required at various levels of the transportation value chain. the fuel economy of E20 is 5% lower than that of conventional gasoline in a conventional car (Gallagher.31 BG (Gallagher.). the efficiency of the cellulosic process could be further increased through technological improvements.3 GL (5. For instance. the currently available technology. with a slowdown in 2008. 2006). 2007). most of which blend ethanol with gasoline in proportions up to 10% of volume (E10)..33 BG) of plants under construction was scheduled in the USA. and to expand into the entire gasoline market. While ethanol production from corn and sugarcane is a well-established process. coal. with Brazil and the USA producing more than 80%. national biofuels blending mandates existed in nine countries. On the other hand. including China. ethanol production has been rising in recent years. 2007). has a limited efficiency of about 35%. for instance. India and France. 7% was synthetic ethanol (produced from ethylene. However. Importantly. should ethanol manufacturing capacity and production rise substantially. All other main trading countries together.. which offsets its octane and oxygen benefits. It has been modeled that. production from cellulosic material is not yet a commercial route. In 2007. In 2007. the large-scale production of second-generation cellulosic ethanol would be essential. including E100. subsidies and tax incentives enacted at the government level. the largest new ethanol capacity (27. By the end of 2006. Dilute acid hydrolysis. about 33 GL was consumed for fuel use and the remainder for beverages and industrial purposes (REN21. Shapouri. Nonetheless. 2006). Growth of the ethanol manufacturing capacity and market is largely driven by blending mandates. 2006).34 Structure of the Bioenergy Business 2.7 GL or 7.1 Status and Potential of Major Biofuels Ethanol In many parts of the world. About 60% of the ethanol was derived from sugar crops. the price effects of ethanol versus gasoline would contribute to increasing ethanol concentration in the market beyond 10% (Gallagher et al.4 2. 2006). Gallagher projected a further doubling of production when all scheduled construction is complete. Hamelink and Faaji (2006) estimated a possible efficiency of 48%. for higher ethanol concentrations. Brazil is to this date the only country where ethanol is used on a large scale and at very high blending rates. flexible-fuel vehicles (FFVs) can operate under a wide range of ethanol–gasoline mixtures and at lower costs. this ethanol concentration does not significantly change car fuel economy characteristics (WSDA. the fuel economy of vehicles declines more than proportionately. More than 30 countries have introduced or expressed interest in programs for fuel ethanol (Rosillo-Calle and Walter. This is due to the lower heat content of ethanol. In order to globally implement transportation fuels containing higher concentrations of ethanol. 2007). and 3% was produced by the bioconversion of other feedstocks (Walter et al. North and South America are the strongholds of ethanol production. 2003. Notably. Almost all of the ethanol produced is consumed domestically. followed by Brazil. which has a planned production increase of 20. However. Of the 45 GL ethanol produced in 2005.4. 30% from grains (mostly corn). The biotechnological significance .

which produces a 93% energy gain as compared to 25% obtained from corn-ethanol (Hill et al. Notwithstanding these observations. US production has increased rapidly to reach about 400 million gallons in 2007. 2007) Notably. biodiesel is renewable. Around 10 other countries have small commercial biodiesel programs. Markets and Policies 35 of this development is perhaps best exemplified by the road map of the US Department of Energy (US DOE.2 Biodiesel Biodiesel is derived from naturally occurring vegetable oils or animal fats that have been chemically modified (esterified) to run in a diesel engine. and maintenance vehicles. and is typically mixed with diesel in a 5% blend (B5). Biodiesel consumption depends. in Germany. 2008). this is a continued trend that is fostered by lower taxes in Europe. including most commercial freight.. making large-scale production possible beyond 2020. thereby making the country the second-largest biodiesel producer behind Germany.The Global Demand for Biofuels: Technologies. Since biodiesel can be refined under normal atmospheric temperature and pressure conditions. 2006). The zero-sulfur content of biodiesel and its solvent and lubricant properties also improve engine performance and lifetime (Johnston and Holloway. Johnson and Holloway (2007) presented a national-level evaluation of potential global biodiesel production volumes and costs. Other countries are currently pursuing similar schemes. Due to their inherent combustion efficiencies and wide array of applications. While Europe’s biodiesel production per year was 20-fold higher than in the US in 2004 (Worldwatch. While gasoline demand is expected to decline by 17. biodiesel in pure form (B100) is used in specially modified diesel vehicles and is available at more than 700 service stations (OECD. 2. construction. has better emissions properties. as exemplified by soybean-biodiesel. and manufacturing processes. distribution infrastructures. it has about 90% of the energy content of conventional diesel. Biodiesel is compatible with existing engines. but it has risen rapidly and dramatically in recent years. which defines a research phase to be completed in five years. Notably. For example. 2005). Biodiesel has a high net energy balance. In some countries. for the time being. global biodiesel production remains small compared with that of ethanol. 66% of the on-road. 2006). the diesel-fleet is projected to rise from 30% in 2005 to almost 43% in 2011 (IEA. diesel engines occupy a significant market segment. and many more have such programs at the research phase.2 GL during the period 2006–2011. Indonesia and Malaysia produce biodiesel for the European market. Moreover. Compared to petroleum diesel. 2007). Their results suggested an . and supports domestic agriculture (Johnston and Holloway. Currently. as demonstrated by a global market growth of 43% in 2007. blends of up to 30% (B30) are used. Vegetable oil used in biodiesel production accounts for only 2% of the global vegetable oil production. The EU and the US together account for over 95% of the global biodiesel demand. with the remainder going primarily to food supply (Johnson and Holloway. liquid fuel demand is covered by diesel. a technology deployment phase within 10 years.4–23. this biofuel can be economically produced across a variety of geographic locations and industrial scales. 2006a). 2007).4. on the existing diesel demand. and a system integration phase of 15 years.

2007). While Indonesia and Malaysia produce at lower than $ 0. These include: (i) the optimization of crop selection.7 per liter cost range. (ii) the growing of dedicated energy crops on marginal lands. 2007). which do not compete for fresh water or farm land (Johnston and Holloway. butanol mixes better with gasoline.4. In the US. India and Mexico it is higher than $0. bio-butanol is a bulk intermediate for chemical synthesis. Malaysia and Indonesia could reap almost 75% of the potential volumes from higher yields. tolerates water contamination. the USA and Brazil are in the $0. 2007). such as palm and coconut. the yields of which are currently much lower than what they could be under optimal sustainable agricultural management.00 per liter. and Brazil – collectively account for more than 80% of the total potential. soybean oil is the feedstock of choice for more than three-quarters of the biodiesel production. Biodiesel cost in much of Western Europe is $0. this practice has severe environmental impacts. On the other hand. and olive oils. animal fats (20%). which makes it more suitable for distribution through pipelines (Ezeji and Blaschek. any vehicle that is able to run on 10% ethanol blends could also use pure butanol. if soybean biodiesel volume targets were to increase by 40% during the 2007–2016 period. feedstock. Johnston and Holloway (2007) estimated that total potential biodiesel volumes could even reach 605 GL per year. and other cost factors in production (Johnston and Holloway. providing a number of possible end products.36 Structure of the Bioenergy Business upper-limit worldwide volume potential of 51 GL of biodiesel from 119 countries. in addition to reducing their CO2 emissions under certain conditions.51–0. palm oil (22%). and undermines the overall carbon balance. coconut oil (11%). including biofuels. . Argentina. however. 2008). and 5% each for rapeseed.29 per liter to over US$ 9. The 12-fold increase is spread over many crops and is mainly attributed to tropical oilseeds. Among the ‘top-10’ producers. The top five countries – Malaysia.89 per liter. sunflower. then soybean prices would be expected to increase by 3. the United States Department of Agriculture (USDA) estimates that. depending on local climate. These two countries are currently growing palm trees for palm oil production through deforestation and land-clearing. However. and (iii) the possible production of oil from algae. 2. Moreover.71–0. distributed over 106 countries.5 per liter cost. Using yields calculated according to sustainable agricultural practices (which are lower than best-case yields). but also cellulose. Consistently. while in China. Biodiesel production costs range from US$ 0.9% (USDA. soybean oil is the cheapest of the vegetable oils in this country. butanol offers further advantages as compared to ethanol. and is less corrosive than ethanol. labor. the United States. thus opening a potentially huge market. While using the same feedstocks and satisfying the same demand for transportation fuels. consequently increasing their GDP per capita and number of jobs.3 Bio-Butanol Similar to bio-ethanol. Notably. the feedstocks most commonly used are soybean oil (28%). Butanol production from biomass could be more energy-efficient than ethanol as some bacteria used in butanol production digest not only starch and sugars. Indonesia. advanced production technologies that could be implemented are those which reduce the impact of biofuel production on global food supplies and improve the sustainability of agriculture production.88. In this scenario.

biomass production from algae in special photobioreactors is being explored as a technology for performing the sequestration of the CO2 originating from fossil fuel-burning power plants. the economic viability of the process remains to be proven. although bio-hydrogen production is technically feasible. Within the framework of this model. indeed. In addition. this could become a huge market in the long term (Peppley. 2007). It is.1 Biofuel Policies and Markets in Selected Countries United States The development of efficient renewable energy sources has been a US policy goal since the first oil embargo of the early 1970s. To balance the growing demand for ethanol.4. individual experiments have demonstrated that this pure fuel can indeed be used (Wiki. since MTBE can pollute groundwater supplies. 2008). 2. however.5. military engagement and high energy costs provided new arguments for investing in biofuel technology as a means to: (i) reduce dependence on oil imports. a market of 11. Markets and Policies 37 Although currently no vehicle is known to be approved by its manufacturer to run on 100% butanol. Consequently. biofuel production and use has grown rapidly since the mid-1990s. as significant technological improvements in fermentative production are required before it can be produced from lignocellulosic feedstocks (OECD. 2006). and (iii) create benefits to the agricultural economic sector. but currently it cannot be predicted when the industrialscale production of bio-butanol will be feasible.4 to 13. driven in the US by federal policies aimed at reducing air and water pollution (INFORMA.2 GL had to be replaced by fuel ethanol (Walter et al. between 2001 and 2006 the USDA made payments of US$ 150 million annually to . As reviewed in Chapters 18 and 19. Interestingly. some industrial partnerships have been formed to develop biobutanol into a viable alternative.5 2. Whether and when this technology will become commercial cannot be predicted. municipal solid waste or waste water. While over two decades of progress has been slow. 2. Fuel cells are expected to be one of the key energy conversion technologies for a transition towards a hydrogen economy (NAE. The US demand for ethanol has been driven primarily by the Clean Air Act of 1990 that made possible the addition or methyl tert-butyl ether (MTBE) and ethanol as gasoline oxygenates. many states had banned its use by the end of 2005. it requires major practical improvements to be economically attractive and to satisfy a potentially very large demand. Moreover. a decentral biogas reformer could produce a hydrogen-rich syngas for fuel cells from agricultural biomass.The Global Demand for Biofuels: Technologies. and could thus be used for hydrogen production (OECD. worth noting that photovoltaic technologies seem to be efficient for energy and electricity generation. 2004).4 Hydrogen from Biomass Using renewable biomass resources could represent a relatively clean and carbon-neutral way to produce hydrogen. 2006). Although this is already technically feasible. 2008). (ii) diminish GHG emissions. Another alternative would be to perform microbial hydrogen production through fermentative and photosynthetic processes in a bioreactor.. However. 2008).

. p. with corn ethanol limited to 15 BG (57 GL) after 2015. Included is a CAFE credit and transfer program among manufacturers and across a manufacturer’s fleet. 2008b). incentives. Likewise. the Volumetric Ethanol Excise Tax Credit (VEETC) of 2004 allows a tax refund of 51 cents per gallon to ethanol blenders on each gallon of ethanol blended with gasoline. 2008b.38 Structure of the Bioenergy Business eligible bioenergy producers under the Commodity Credit Corporation Bioenergy Program to encourage increased purchases of agricultural commodities for expanding production of biofuels and to encourage the construction of new production capacity (INFORMA. the US Energy Policy Act of 2005 established a national Renewable Fuels Standard (RFS) which creates incentives for biofuel production and use. the Small Ethanol Producer Tax Credit provides a 10 cents per gallon production income tax credit for the first 15 million gallons to small ethanol production facilities with a productive capacity of less than 60 million gallons. where it advocated in the 2007 release the implementation of policy measures to advance biomass technologies and the biobased industry (www. In addition. Credits are 50 cents per gallon for biodiesel (monoalkyl esters of long-chain fatty acids derived from plant or animal matter) and US$ 1. including light trucks.9 GL) of biodiesel by 2012. and an additional 5 Bg (18. an increase by 30% as compared to the 2008 average (NHTSA. A new Corporate Average Fuel Economy (CAFE) standard for passenger automobiles. A mandatory RFS of at least 36 BG (136 GL) of ethanol per year by the US President announced an increase to 133 billion liters (35 BG) of renewable fuels by 2017 – that is. 2008). the Department of Energy’s biomass research and development initiative releases an annual Roadmap for Bioenergy and Biobased Products in the United States.6 GL) of cellulosic ethanol by 2022.4 cents per gallon. Furthermore. For comparison: according to the Annual Energy Outlook 2008 (EIA.5 million per year and per producer (INFORMA. capping the federal tax credit to a maximum at $1. which funds grants and loan guarantees to agricultural producers (farmers and ranchers) and any small rural businesses.4 cents per gallon In addition to these direct incentives. In addition. 2009).4 cents per gallon. Moreover.18): . as exemplified by the fact that in the 2007 State of the Union addresses.usda. the 2007 Energy Independence and Security Act (EISA) established specific tax credits. .00 per gallon for agri-biodiesel (biodiesel derived solely from virgin oils and animal fats) and renewable diesel (diesel derived from biomass using thermal depolymerization) (CULS. diesel at 24. 2006.6 billion increase in renewable energy funding and a US$ 2. and supports research on new biofuel technologies and cellulosic feedstocks. at least 16 BG ( What is more. The impetus to drive the implementation of biofuels is expressed at the highest levels of the US economic and technology policies. 2006). of 35 mpg by 2020. gasoline is assumed to be taxed in the USA at 18. that is. the 2002 Farm Bill established the Renewable Energy Systems and Energy Efficiency Improvements Program. The goal is to encourage small-scale producers (such as cooperatives) to start ethanol production. the 2007 Farm Bill proposal recommends a US$ 1. while US$ 500 million would be made available for bioenergy and bioproducts research (www. and kerosene jet fuel at 4. including the following (EIA. nearly five times the 2007 level.brdisolutions. or standards for promoting the implementation of biofuels.1 billion loan guarantee program. RFA 2006b).

Notably. Markets and Policies . The top five producing states (Iowa.eere.2 GL/year) (RFA. 2008b). or 83% in 2030. the biofuel component of motor fuels is projected to grow substantially while the fossil fuel content of gasoline and diesel is expected to decline from 515 GL (136 BG). to 473 GL (125 BG). In addition. this profitability started to significantly erode in 2007 when production capacity and corn price concomitantly increased. as it essentially disqualifies any future corn ethanol production facilities from using coal for process heat. or 96% in 2006. the 147 ethanol biorefineries existing in the US had a demonstrated total production capacity of 32. in April 2008 many states have set a Renewable Portfolio Standard. 39 A life-cycle GHG standard of 20% emission reduction for corn-based ethanol. after which the ethanol requirement is expected to be met by the increased consumption of E85. According to the Energy Information Administration (EIA. thus causing at least in part a sharp decline in profit rates (Crauss. is also the world’s largest and fastest growing consumer of fuel ethanol. Illinois. More ambitious mandates have also been considered. despite the dynamism of this nascent industry during the first decade of the twenty-first century. The RFS ethanol production capacity is expected to double from 2006 to 2012. in nine years. 2008).3 GL in 2030 (EIA. 2006a). For instance. in 2005 the state of Oklahoma passed a tax credit of 20 cents per gallon for biodiesel facilities. including biomass (www. however. This is an important decision. whereas fuel ethanol consumption is expected to grow almost fourfold in the period 2005–2030. As a result of these and other policies. based on the 2005 emission level. Notably. Minnesota) together represented 80% of the online production capacity in early 2006 (16. Due to the rapid growth in the largest consumer of motor gasoline in the world. South Dakota. As a result. the United States. 2007) and in investments in green-field manufacturing projects for corn ethanol production (English.08 BG) of additional manufacturing capacity (RFA. and on the other hand to force automakers to cut CO2 gas emissions by 30% in new cars and light trucks by 2016. future developments in cellulosic ethanol manufacturing plants. In April 2008. with short payback periods. 2007). these initiatives were denied by the Environmental Protection Agency (Broder and Barringer. Similarly. the US market for E10 will be saturated by 2014. financial return rates in the bioethanol industry initially soared. with regard to biomass for electricity production. 2006). the country has reached an almost fourfold increase in production capacity. representing 19. many states provide incentives for converting biomass into energy to stimulate demand or to help the establishment of new biofuels producers.2 GL (8. California has established a Low-Carbon Fuel Standard of 43 mpg to achieve on the one hand a reduction of at least 10% in carbon intensity of transportation fuels (Farrell and Sperling. which implies that a certain percentage of a utility company’s overall energy capacity or energy sales must be derived from renewable resources. a handful of individual states have tried to outpace the federal government in moving biofuels forward and reducing GHG emission. Taking the lead. 2008). such as reaching annual production or consumption levels of 60 BG of ethanol (227 GL) by 2030 – provided that cellulosic ethanol becomes a feasible alternative (Foust.52 BG) per year. reaching 55.2 GL (5. 2006). also depend on the evolution of the market for flexible fuel vehicles. Advanced biofuels are defined as renewable fuels that reduce emissions by at least 50%. 2006). with a maximum annual payment of US$ 5 million (EESI. Nevertheless. 55 such refineries were under construction and six under expansion. . Similarly. Nebraska.The Global Demand for Biofuels: Technologies. 2007).

40 Structure of the Bioenergy Business Particularly. and export demands.5. 2. or agricultural and forestry practices. Since October 2003. 2007): . High oil prices and the ratification of the Kyoto Protocol in 2005 have provided additional incentives to strongly promote the use of alternative fuels. fuel ethanol production in the US will in the long run depend on the use of cellulosic materials as primary feedstocks. Within the 2015 time frame. The objective of this policy is to double the share of renewable energy sources in the EU. 2006. Such an amount is sufficient to meet more than one-third of the current demand for transportation fuels while still meeting food. 2006). With a set of around 20 actions. individual member countries have implemented different policies and measures to reach their respective targets. as well as biofuels mix strategies that optimize the benefits to each country. 2008. without the disruption of agricultural markets (INFORMA. This biomass resource potential can be produced with relatively modest changes in land use. the EIA (2006) has estimated that by 2030 about 10% of the total sales of new light-duty vehicles will correspond to flexible-fuel vehicles. This set an overall target of 2% biofuels in the fuel transportation mix of the EU by 2005. A main driver for biofuel production in the EU has been the Green Paper “Towards a European strategy for energy supply” (EC. 2001).2 European Union Like the United States. On the other hand. over 1. A number of policies have expanded the use of biobased fuels (see INFORMA. from 6% in 2005 to 12% by 2010 – that is. such as a change to the production of perennial grasses like switchgrass or miscanthus (Khanna et al.. (2006) have calculated. The European Commission (EC) Biomass Action Plan of December 2005 promotes the sustainable use of biomass as a key part of the EU’s future energy strategy. As Perlack et al. provided that environmental preservation concerns are met. the EU aims at reducing its dependence on external energy sources. the EC attempts to increase the transformation of various biomass feedstocks into energy. and a target of 5. However. The US biofuel industry has been historically confident that domestic agriculture can deliver the current production mandate by 2012. and another 10% will correspond to hybrids. from 69 million tons of oil equivalent (MTOE) in 2003 to around 150 MTOE by 2010. These measures are estimated . although this remains unlikely in the absence of any significant government funding (Hofstrand. 2008. Given the limits of corn ethanol. which provided the fundamental basis for the European Biofuels Directive (2003/30/EC) of May 2003.. Walter et al. Scheffran and Bendor 2009). such as feedstocks derived from forestry or agriculture as well as waste materials. the Directive on the Taxation of Energy Products has allowed the tax exemption of renewable fuels in any country of the EU. 2008). much underutilized land could also be put to contribution for biomass production.3 billion dry tons per year of biomass from forest land and agricultural land alone are potentially available in the US. and at creating a new stimulus for the rural economy (Faaij. such as cropland managed by the Conservation Reserve Program (CRP) that could supply such natural raw materials. commercial cropland conversion from traditional crops to dedicated biomass crops is possible. . Tyner. 2006). feed.75% by 2010.

2008) Among the largest European biodiesel producing countries.89 MT of biodiesel in 2007. 2008). and 1% of the transportation fuel market share in 2003. 2004. via implementing economically improved agricultural practices. 2006). biodiesel accounted for 76% of the biofuels consumed in the EU.” (EU. Despite significant growth rates.The Global Demand for Biofuels: Technologies. The 10% mandate is “. the European Commission presented its new Directive for Renewable Energy as an integrated proposal for climate action. . 0. A major limiting factor in converting a large diversity of feedstocks into ethanol is the costs of these feedstocks themselves.5% in 2008) and a 10% minimum target for the market share of biofuels by 2020 (EC. while corn and potatoes have also been used. secondgeneration biofuels becoming commercially available and the Fuel Quality Directive being amended accordingly to allow for adequate levels of blending. The Strategy for Biofuels. Germany manufactured 2. Between 2002 and 2007. showing the difficulty for EU producers to compete with unfair B99 imports from the U. implemented in February 2006. the average biofuel contribution remains small in the EU (respectively.e. . The predominant feedstocks used for ethanol production in the EU are sugar beet and wheat. Biodiesel production increased from 4. several countries are still lagging far behind in implementing either biodiesel or bioethanol (Rosillo-Calle and Walter.S. .87 MT. and following repeated complaints that US subsidies for B99 biodiesel are in breach of World Trade Organization rules.7 MT in 2007. The total annual subsidy for biofuels provided by EU governments reached e 3. 2008a). 2006). Because of the constraints of fuel ethanol production with regard to current available technologies and feedstocks. includes market-based. the capacity of fuel ethanol production in Europe by 2008 was estimated as 16 MT. including tax exemption and compulsory biofuel blending specific for each country. to have the potential to reduce CO2 emissions by 209 MT per year.7 billion in 2006. However. In 2007. Research funding supports cost-effective and environmentally friendly methods to mass-produce ethanol. legislative and research measures to boost the production of fuels from agricultural materials. 2006). Nevertheless. 2008). By the year 2030.5. in addition. the total biodiesel production in Europe grew by more than fivefold (EBB. negative change in market conditions in 2007. and to create up to 300 000 new jobs in the agriculture and forestry sector (INFORMA. the European Biodiesel Board (EBB) expressed its view that this observation highlights the “. cellulosic) biofuels.8%. subject to production being sustainable. 2008). such costs may be reduced in the short to mid term by way of implementing cellulosic ethanol or. On the other hand. . This was a considerable decline in growth rate as compared to the 65% that occurred in 2005 and 54% in 2006 (EBB. In a statement that exemplifies the exacerbation of global competition for the renewable fuels market. and 2005). to diminish crude oil imports by 8%. The directive sets an overall binding target for the European Union of 20% renewable energy by 2020 (from 8. the EU also is placing a large effort on the development of second-generation (i. 0. one-quarter of the petroleum consumed in the EU is to be replaced by biofuels (BIOFRAC. which represents a yearly growth of 16.6. .” (EBB. By comparison. On 23 January 2008. surpluses of wine have also been converted into ethanol. Many countries . more simply. which is probably an underestimate. ethanol production from food crops such as corn and cereals remains noncompetitive in the EU when compared to gasoline and diesel (Faaij.9 MT in 2006 to 5. Markets and Policies 41 . followed by France with about 0. 2006).

Consequently. sugar prices rose and oil prices fell. because of high sugar input prices and initial excess capacity effects. 149). 2008). Total number was about 1000 new vehicles per year in 1997–1998. Fischer–Tropsch liquids or hydrogen (Faaij. It is also worth noting that Brazil’s ethanol exports grew by 8% from 2004 to 2005. Notwithstanding the strong political will demonstrated by the EU to implement biofuels on a large scale. 2008). Fuel ethanol demand began to grow significantly again in Brazil after 2001. this deregulation beneficially transformed ethanol production and exports into entirely market-driven economic activities. In light of this debate. The tendency demonstrated by biofuel manufacturers in the European market is to avoid the large-scale implementation of biofuels until feedstock costs are significantly reduced. 2008). almost in parallel. that is. 2006). While some governments have begun rolling back subsidies for biofuels. Notably. The main objective of this program was to encourage the use of ethanol in domestic transportation fuels and to diminish the dependence on oil imports. Brazil has been the largest producer of biofuels over several decades.3 Brazil Privileged by abundant and cheap sugarcane feedstocks. especially those grown on vulnerable lands (NYT. and increased the capacity of the vehicle fleet to consume ethanol (INFORMA. To this date. the EU is considering a ban on certain types of biofuel. However. Due to concerns over the global food crisis and the sustainability of biofuels. with the amount being determined each year. Brazil experienced serious ethanol shortages in the early 1990s. The Brazilian experience with ethanol–gasoline blends dates back to the 1930s. when in 1998 the price of ethanol was liberalized. However. p. the EC seemed in April 2008 to back away from its insistence on imposing a 10% quota of biofuels by 2020 (Traynor. methanol. Brazil paradoxically imported substantial volumes of ethanol to reduce its supply constraints. mostly as a result of increased exports to Japan and the EU. subsequent to which Brazil produced in 2007 a total 18. During this period. 2006). Brazilian environmental policies require gasoline to contain specific amounts of alcohol. consequently. When. The high oil prices experienced by the global economy in 2005 generated a renaissance of ethanol production in Brazil. 2008.9 GL (5 BG) of ethanol (RFA.5. the sales of pure ethanol-fuelled cars reached 92–96% of all cars during the 1980s. dimethylester (DME).42 Structure of the Bioenergy Business of the EU are also investigating alternative routes based on biomass gasification for syngas production and conversion to biofuels. but it was the oil shocks of the 1970s that constituted a genuine tipping for Brazil. during the late 1980s. the . others would favor high import tariffs so that the European biofuel industry could implement a dynamic biofuel value chain starting from the production of biomass feedstocks in the EU. Brazil regulated the price of ethanol relative to gasoline. invested in ethanol distilleries. sales of ethanol cars began to rise from the launch of flex-fuel vehicles (FFVs) in 2003. the market penetration in Brazil of these new vehicles reached about seven million (25% of the vehicle fleet) in 2008 (Zuurbier and van de Vooren. subsidies for ethanol production were gradually eliminated. making projection on the long-term biofuels market in Europe remains an uncertain exercise. which reacted to the oil shock-induced economic crisis by implementing the PROALCOOL program. and to significantly contribute to the reduction of GHG emission. 2.

2007). 2007). sugarcane production has become the growth engine of the Brazilian ethanol industry.The Global Demand for Biofuels: Technologies. including biofuels (METI.5. in 2005. by 1985. During the period April 2005 to May 2007.3 billion liters.9 and 3. 2008). It is estimated that. and scheduled for completion by 2012 (Gallagher. Particularly. 2006a). As a result. and there is significantly more potential in the future (Shapouri et al.. Japan has proposed a target of using in its energy mix 0. ownership and operation of vehicles supplement the tendency for Japanese consumers and car makers to shift towards smaller and more efficient vehicles. most probably with FFVs (Ethanol News. Taxes upon the acquisition.13 BG) of biomass-derived fuels by 2010. 2. about US$ 11–12 billion had been invested to create the infrastructure in Brazil to produce 16 GL per year (Walter et al. Japan’s long-term energy supply/ demand outlook released in March 2008 projects biomass energy consumption in FY 2020 at between 2.. the Brazilian ethanol production capacity is expected to more than double from 2006 to 2015 (Walter et al. 2006). Markets and Policies 43 expansion of Brazilian ethanol exports have since slowed down. It is expected that in this country gasoline demand would grow by less than 0. while transportation energy use would drop by 0. 2008). Japan was the second-largest importer of ethanol. The Japanese government also proposed an E3 blend standard (about 1. Brazil has continuously increased it ethanol production. while the remainder would be joint sugar/ethanol plants.5% annually between 2006 and 2011 (IEA. most of which was used as fuel ethanol. and Taiwan) has a fully developed biofuel strategy or a significant feedstock and production capacity that could satisfy their demand in transportation fuel. Although this country is a producer of synthetic ethanol. 2007).4% per year on average in the period 2003–2030 (EIA. The Japanese industry as a whole is playing a major role in developing advanced technical solutions. Over recent years. Alternatively. . Japan has implemented policies to promote the large-scale use of fuel ethanol or ethyl tert-butyl ether (ETBE).47 BG) in 2004 as a step towards a national E10 standard by 2010 (INFORMA. which is about 1% of the projected fuel use. 2008). 2006). One million jobs are directly associated with the Brazilian ethanol industry.. 2006c). it has largely insufficient natural biomass resources to produce ethanol to the necessary scale.78 GL or 0. Targeting at improving its energy security and meeting its Kyoto targets. The Japanese Environment Ministry has set the goal to have all cars in Japan capable of running on ethanol by 2030.5 GL (0.4 Japan None of the developed countries in Asia (Japan.Today. a total of 126 plants was planned or were under construction. to 4491 million gallons in 2006 and 5019 million gallons in 2007 (RFA. 2007). As a result. 16 major Japanese firms–in cooperation with Japan’s top universities and government agencies–plan to develop technologies to mass-produce low-cost bio-ethanol fuel from agricultural and industrial bio-waste (Japan. 43 are planned to specialize in ethanol production from sugarcane. Japan is one of the main consumers of motor gasoline in the world. and is heavily dependent on imported oil. South Korea. In order to contribute to the fulfillment of its commitments to the Kyoto protocol. the use of an ETBE blend is seen as a valid option by part of the industry (Nippon. and following a top-down approach initiated by the Ministry of Enterprise Trade and Industry and the Ministry of Education. 2006). Of these plants.

more than three times as much as China which is third (FAOSTAT.5 GL (INFORMA. Developing countries are increasingly replacing petroleum imports with biofuels. Weather effects can be strong in India. China has set the target of producing from renewable sources by 2020 a total of 11 MT of biofuels (INFORMA.. However. in the tropical and subtropical regions the costs for raw materials are generally much lower than in industrialized countries (Shapouri et al. 2006). At the Renewables 2004 conference in Germany. 2009). Malaysia and Indonesia are low-cost producers of palm oil (Reinhardt et al. although they often lack the resources to provide subsidies and other tax incentives to promote biofuels at a larger scale. The projected fuel ethanol demand in India in 2010 is 1. and in spite of a large energy demand. fuel ethanol is exempt in this country from consumption tax and value-added tax. and only a few small plants are operating that mainly use waste cooking oil or oilseeds as feedstock (INFORMA. (2007) concluded that in sub-Saharan Africa. adequate weather conditions.3% over the period 2006–2011. the Caribbean and Latin America.5 Other Countries As the Brazil case demonstrates. there is a large potential for bioenergy production due mostly to one factor – the availability of agricultural land. Being the second largest producer of ethanol in Asia. 2007). ethanol production from sugarcane has been stalled in August 2005 (INFORMA.6 2. such as jatropha. thus creating a strong demand for alternative transportation fuels (IEA. Because of serious sugar shortages. 2008). 2006a). as exemplified by the economic impact of the 2005 drought that resulted in a low sugar crop supply.44 Structure of the Bioenergy Business 2. India is expected to expand its use of road transport fuels by 5. Many developing countries have a good potential for biofuels production due to the availability of underutilized land.. and economic uncertainties. 2006). 2006). food crops are a priority for land use in China. 2006). the use of biofuels can both reduce expenses and strengthen energy security. several provinces have introduced compulsory ethanol-blended gasoline. 2006). The biodiesel program is less developed than the bioethanol program. decisions and assumptions which are affected by political. technical. Since many developing countries are net oil importers.1 Perspective Future Biofuel Projections and International Trade Future biofuel demands depend on a number of variables. India is also the world’s second largest producer of sugarcane (358 MT in 2008). for mixing in diesel (Jatropha World. while Taiwan and South Korea have already started biodiesel trials (INFORMA. and thus to increased feedstock prices and ethanol imports.5.6. It is also worth noting that the Indian government has implemented a biodiesel purchasing policy to make public sector oil firms purchase vegetable oil extracted from plants. Scenario analysis is a . 2006). Facing a rapidly growing demand for transportation fuels (with an expected consumption of 228 MT in 2020). Notably. and the availability of a cheap labor force. China announced the national commitment of obtaining 16% of the country’s energy from renewables by 2020 (Martinot and Junfeng. moreover. A study by Smeets et al. 2009). 2.

through international trade that is not distorted by barriers or tariffs. usually assuming a certain set of policies. thus promoting the specialization of particular manufacturers to products for which they have a comparative advantage. sugar or oilseeds can compete with imported petroleum above a threshold price which varies across regions. EU 25) to the developing countries. and European ethanol at more than US$ 80 per barrel (Inter-American Development Bank. displacing 10% of the fossil fuel as calculated from their estimated demands. According to a report of the Inter-American Development Bank. This relies on the assumption that more than 60% of fuel ethanol consumption in 2030 is expected to be concentrated outside of the developed countries. Within 25 years. consumers would be able to fully benefit from market forces and thus would be free to exert their choice to switch to those providers that produce biofuels or sustainable commodity chemicals at the lowest cost or highest quality. The trend will favor development of corn and wheat ethanol capacity in North America and Western Europe. 2008). Japan. the international trade of biofuels has remained limited (Table 2. which is based on ethanol mandates and historic data of motor gasoline consumption in the US.” (Freedonia. most of which is undenatured ethanol. utilizing and trading crops for both food and fuels connects energy and food markets and affects crop price on a global scale. The estimated results for motor gasoline and ethanol consumptions in 2030 compared to the baseline year 2005 are reported in Table 2. . China. . This price differential creates incentives for an ethanol flow on the global market from Brazil to the USA. Nevertheless. Markets and Policies 45 technique that helps one understand possible futures. with potential undesired effects.4).3. this not only adds to costs but also increases the environmental impact of biofuels. including carbon emissions. Japan. Moreover. In 2006. EU 25. fuel ethanol demand would reach 272 GL in 2030 (an almost ninefold increase from 33 GL in 2005). rapeseed oil in Europe. Due to increased efficiencies. (2007) provides a global ethanol market forecast. US ethanol at US$ 60 per barrel.” Similarly. and as a result a substantial biofuel manufacturing capacity needs to be established in developing countries. international trade requires transportation over large distances. imports are becoming a significant factor to satisfy the growing demand for biofuels. a study by Walter et al. without adequate sources of domestic supply. and increased global biodiesel demand. Notwithstanding this advantage. Brazilian ethanol is already competitive at a petroleum price of US$ 40 per barrel. To date. ethanol trade has doubled between 2000 and 2005 to reach about 13% of the estimated world ethanol production of 45 GL. and palm and jatropha oil in the Asia/ Pacific region. This report concludes as follows: “Market expansion will come from a more than doubling of the world market for ethanol. 2007). In the baseline scenario. most of the conventional gasoline consumption will likely shift from the most developed countries (USA. Brazil and in the rest of the world. and from there to Europe. OH). world demand for biofuels will expand nearly 20% annually to 92 million metric tons in 2011. According to an April 2008 study by the industry research firm Freedonia Group (Cleveland. Also shown are ethanol targets and growth rates. Next generation cellulosic ethanol and algal biodiesel technologies will become commercially significant in the longer term.The Global Demand for Biofuels: Technologies. the . integrating such factors. For numerous countries. as well as sugar-based ethanol production in South America. biofuel production processes based on grains. the “. Biodiesel production will center on soybean oil in the Americas. The demand–supply gaps of countries constitute a useful indicator for assessing potential international biofuel trade volumes. Notably.

8%) 60.5% 13.3% 13.0%) 53 (5. Structure of the Bioenergy Business USA 1. 55.46 Table 2.6%) 746 (38.3 GL by 2030 2.6 GL by 2020 (E10) E1 by 2010.5%) 61 (5.3%) 50.6% 15.5% by 2010.3 (3.63 China 6 4.3 (3. E10 2.3 (40.4% 8.7%) 126 (6.3%) 33 272.0%) 166 (8. Adapted from Walter et al.3%) 1.2%) 9.62 1.9%) ETH annual ETH annual growth rates growth rates 2005–2010 2005–2030 8.1% 5.5%) 1213 43 (2.8% 5.5 GL by 2010.3 World gasoline (GAS) and fuel ethanol (ETH) consumption 2005–2030.4%) 21. 12.4 8.8%) ETH targets/ GAS Growth GAS rate (%) consumption consumption estimates (GL) 2030 2003– (GL) 2005 2030 est.4%) 55.5 (1.5%) 164 (13.0%) 1.2 (36.2% 12.6 (7.6 (4.76 27 GL by 2012.4% 100.0 (3.5 0.0 (18.75 1.4 EU-25 À2.1 Rest of World minus Brazil Brazil World 18 (1.2%) 1924 — — 3.19 Japan 0.8% .1% 19% Country/ region Growth rate (%) 2006– 2011 est. (2007) ETH ETH consumption consumption (GL) 2030 (GL) 2005 15.9%) 36.7%) 71 (3.3 (46.80 13.3 (20. 20.91 3.9%) 0.4% 26% 34.0 (13.5%) 1.2%) 378 (31.5 1.2%) 771 (40. 528 (43.26 2.3% 20.0% by 2030 E10 2015 onwards 2.71 À1.

906 0. On the other hand.043 0.016 0. neither do they apply to participants to the Caribbean Basin Initiative (CBI) agreement and the Andean Trade Preference Act.02 0. and thus the import needs of the major importers. Gallagher. It is remarkable that in 2006 the European ethanol imports represented only a small fraction of the EU’s 17.The Global Demand for Biofuels: Technologies.042 0. Markets and Policies 47 Table 2. Ethanol imports to the USA and Europe are subject to import rules and tariffs. Copyright 2007 John Wiley & Sons Ltd Exporters US Brazil China Caribbean Other Total imports 0.702 EU 0.7 US$ mÀ3) plus a 2.06 0.269 BG). exporting one-quarter of its 16. equivalent to about 9% of the global ethanol production.126 0.091 1. 2007).209 Total exports 0.197 BG) (Gallagher. 2007).W.58 GL (0. 2007).016 0. Bioproducts and Biorefining. and Pacific countries.7 GL). Caribbean. world trade in 2012 would be about 7.00 0.46 BG).4 World ethanol trade for 2006 (in billion gallons). Walter et al. 92% of which were exported to a dozen countries (Walter et al.5% ad valorem tariff on ethanol.00 0. Counting the total sum of the estimated 2012 deficits.179 Japan 0.906 BG). 2006.5 GL ethanol produced in 2005 from sugarcane (Valdes. Under the MFN regime (which includes Brazil).5 GL (1.08 0. import duties do not apply to Canada.. of which 0.179 BG) and Japan (0. China (0. The major importers in 2006 are the US (0. Smaller amounts were imported from China and other countries.197 0.463 worldwide ethanol trade volume was about 5. 2007). 2006b). Up to 7% of the current US production is duty free (Gallagher et al.154 0.4 GL (4. of which about 1.68 GL (0. In order to offset lower production costs in other countries and to impose a barrier to imports as a means of promoting the development of a domestic bioethanol industry. while the major exporters are Brazil (0. This amount represents approximately 50% of the 2005 world exports.. Reprinted with permission from P.00 0.6 BG) consumption.206 Other 0. 2007).110 0.099 0. the EU (0. the US imposes most-favored nations (MFN) import duties of 54 cents per gallon (142. To enable biofuels from Least Developed Countries (LDCs) to enter the European market both duty.and quota-free.03 0.00 0. In 2007.467 0. and a duty of e102 mÀ3 on denatured alcohol.5 BG. and the Caribbean countries (0. Within the Generalized Systems of Preferences (GSP). Israel.269 0. 2008). duty-free status was applied to 79 African. thus demonstrating the narrowness of the nascent international biofuel trade.177 0. and Mexico. the EU imposes a duty of e192 mÀ3 on undenatured alcohol. 103–118.. and as a result 26% of the imports had no duties (Walter et al.7 GL were imported directly from Brazil and much of the rest from Caribbean countries.016 0.. 1. Europe has . A market and policy interpretation of recent developments in the world ethanol industry. The EU imported 0. This hurdle is in addition to the domestic Federal tax exemption of 52 cents per gallon (Elobeid and Tokgoz.18 BG) of ethanol in 2006.15 BG) originated from Brazil. By far the largest ethanol exporter in recent years has been Brazil.133 Importers Caribbean 0.007 0.702 BG). the USA imported more than half the ethanol traded worldwide (or 2.133 BG). which serve as trans-shipment stations for duty-free ethanol imports to the US (OECD. Biofuels.

“Africa has the largest bioenergy potential in the world”. has a huge potential for biomass production. there is broad agreement that Africa has significant opportunities to use biomass for energy development to displace fossil fuel and enhance energy access. If well implemented.9 GL (38% of the estimated consumption). Converting this volume of edible oil into biodiesel would dramatically affect food supplies and increase feedstock prices across various economic value chains. The Biopact is a Brussels-based connective of European and African citizens who strive towards the establishment of a mutually beneficial ‘energy relationship’ based on biofuels and bioenergy (http://news. the biofuel industry could be a major growth sector for these countries. removing the barriers imposed on biofuels trade is not specifically part of any international trade negotiation (Hazell and Pachauri. 2008). including regional energy projects (UN Foundation. unless driven by a strong domestic demand and efficient domestic markets in biofuels. According to Smeets et al.48 Structure of the Bioenergy Business preferential trade agreements with LDCs under the Everything But Arms (EBA) initiative. fuel. the European Investment Bank (EIB) opened regional representation for West Africa and the Sahel region in 2005. and offer prospects to investors who could import the technology for transforming crops into fuel. Walter et al. the US and EU trade policies exert a significant impact on global biofuel production. a fair North–South biofuels trade that is economically beneficial for both sides may nevertheless be realized. North–South cooperation among . Although neither the US nor the EU can domestically produce sufficient amounts of renewable fuels to meet their own long-term policy targets. concluded their 2007 study as follows: “In case USA and EU set quotas equivalent to 30% of their estimated consumption of ethanol. progress has been hampered by the shortage of any infrastructure to cope with biofuel production. It is estimated that Brazil alone could supply this demand by 2030. foremost for domestic consumption but also for export. and particularly in developing countries. 2006). the production of biofuels after food. the EU-Africa Infrastructure Trust Fund Agreement was signed to support regional infrastructure projects in sub-Saharan Africa.mongabay. However. that is. Africa. Regarding future ethanol trade. and the export potential that these represent for biofuel producing countries. but other countries in the world – mostly developing countries – have the potential to be large-scale producers and exporters of fuel ethanol during the following 25 years. (2004). To improve the climate for and increase the amount of energy investments. large investments in biofuel production capacities in developing countries remain unlikely. However. imports would be increased to 45. and fodder needs for local populations and livestock have been satisfied – and without deforestation. Although there is some dispute in the expert community over how large that potential really is. In 2007. it is clear that in the absence of any relaxation of trade constraints such as tariffs and taxes. Certain LDCs have land suitable for growing biofuel crops. Given the sizes of the US and EU ethanol and diesel markets.” The biodiesel export potential identified by Johnston and Holloway 2007 represents a theoretical 21-fold increase over the current production. not all of this potential could be realized. given possible comparative advantages of biomass productivity and efficiency in developing countries (Hazell and von Braun. and particularly sub-Saharan Africa. Notwithstanding this hurdle. 2006). Nonetheless. and to reinforce the development of renewable energy in Africa. since the necessary feedstocks would represent up to almost one-third of all vegetable oil demand.

will reduce biodiversity and may even cause greenhouse gas emissions rather than savings. bring significant income and sustainable development in rural areas (John and Watson. if biofuels were to be produced in a socially acceptable and fair manner. the OECD suggests that the mediumterm impacts of current biofuel policies on agricultural commodity prices are important. not considering the potential impact of these increases on food and feed prices and availability. due to biofuel demand. 2006). assuming the full implementation of the 2007 US EISA and the new EU Directive for Renewable Energy. Notably. would aim not only at establishing ecological and social standards instead of trade barriers – thus enabling the global economy to benefit fully from comparative advantages effects – but also at opening fair market access and implementing sustainability standards for tropical biofuels – thus driving the economic development of numerous countries. and increase access to modern energy services” (UN Foundation.g. The International Food Policy Research Institute (IFPRI) predicts that an aggressive biofuel scenario – without concomitant technological breakthroughs that would dramatically increase productivity throughout the biofuel value chain – could lead to significant price increases for some food crops (von Braun and Pachauri. without having any significant impact (OECD. biofuels have the potential to alleviate poverty. 2. the biofuel industry could. In its 2008 report ‘Economic Assessment of Biofuel Support Policies’. up to 12% of global coarse grain production and 14% of global vegetable oil production could be used for biofuels. . For example. by its very nature. as suggested by Mathews (2007).The Global Demand for Biofuels: Technologies. these numbers could rise to 20% and 13%. respectively. 2008). reduce reliance on imported oil. on food price. In June 2005. This is because the displacement of existing agricultural production. The report concludes that “. the United Nations Foundation launched the Biofuels Initiative aimed at promoting the sustainable production and use of biofuels in developing countries. 2006). the demand and political support for biofuels has been declining in parts of the world. A careful analysis is required to assess the advantages and disadvantages of large-scale biofuels production (Hazell and von Braun. Notably. . as exemplified by the suspension of the World Trade Organization agriculture negotiations in Doha in July 2006. . land use change.6. to this date the lowering of trade barriers on agricultural products – including biofuels – has not yet been solved. 2007). implementing a ‘Biopact’ for a North–South Trade in biofuels. 2006). . create sustainable rural development opportunities. is accelerating land-use change and.. respectively up from 8% and 9% in 2007. if left unchecked. The introduction of biofuels . there is a future for a sustainable biofuels industry but that feedstock production must avoid agricultural land that would otherwise be used for food production. but should not be overestimated. carbon and energy balance). According to the same report. Furthermore. the EU has adjusted its mandates for biofuels and has established conditions and criteria for the sustainable use and certification of biofuel (Directive EP 2008).2 Biofuel Impacts and Debates Due to the growing debate since 2006 on the potential adverse impacts of biofuels (e. A comprehensive assessment of the indirect impacts of biofuels has been prepared by Gallagher (2008) which represents the review adopted by the UK Renewable Fuels Agency. According to the UN Foundation “. Markets and Policies 49 countries and companies appears to be essential for building costly biofuel infrastructures. Notably.

Available at www. Crauss.P. http://europa. Energy Information¼41189. rising oil prices by replacing gas cars with ethanol 2007. European Commission. EIA (2008b) Annual Energy Outlook 2008. www. Luxembourg. research into second-generation biofuels. A slowdown will also reduce the impact of biofuels on food commodity Barringer. Biodiesel and renewable diesel used as fuel.htm. E. New York European Biodiesel Board. J.eia. A. in addition to the development of advanced technologies to improve productivity. US code collection. Report of the Biofuels Research Advisory Council. 2006. CULS. OECD: Paris. R. Cornell University Law School.eia. such as cellulosic ethanol or butanol. www.Removal of U. Biofuels in the European Union – AVision for 2030 and Beyond. http://www. are essential goals that must be achieved in the near future if the ‘biofuel vision’ is to become reality. Biofuels: Is the cure worse than the disease?. Elobeid and S.doe. and Brazilian ethanol markets.html. EBB. 2008.pdf. http://www. EC 2001. New York Times. Broder. www. Environmental & Energy Study Institute. 11–12 September. World Biofuels Production. Japan to fight global warming.cornell.europa. Available at www. R.iastate. or biodiesel produced using sustainable agricultural practice. Tokgoz. http://ethanol-news. 27 May. Round Table on Sustainable Development.PDF. Available at www. 20 December.M. http://www. September 1999.S.” As a result. Ethanol News. Corn ethanol plant 2005 Year in Review: U. Working Paper 06-WP 427 2006.sugarcanebioethanol.renewableenergyworld. Sugarcane-based bioethanol:Energy for sustainable development.newslib. EU Energy Ministers Approve Binding Biofuels Mandate. English. US Department of Energy. Unstudied Risks-Economic Assessment of Conversion From MTBE to Ethanol in California.html. europa. 2007 – 2008 Production statistics show restrained growth in the EU due to market conditions and competition from U.htm. Prepared for the American Methanol Institute. Promotion of the use of energy from renewable sources. References AMI.S. 23 January Structure of the Bioenergy Business should be significantly slowed until adequate controls to address displacement effects are implemented and are demonstrated to be effective. Towards a European strategy for the security of energy supply. press 4 January. US Department of Energy. 2006. notably oil seeds. BNDES. BIOFRAC. accessed January 16. http:// Green Paper. MONITO1. Coordination BNDES and CGEE – Rio de Janeiro. (accessed 16 January 2009). 2007. says 17 States can’t set emission rules. Annual Energy Outlook 2007. European Commission. B99 imports.html. 2008. Tri-Cities Business Rev. which have a detrimental effect upon the poorest people.ebb-eu. ethanol domestic and trade distortions: impact on U. 30 September.calgasoline. 06/25/2008. Energy Information Administration. EESI. EIA (2008a) International Petroleum (Oil) Consumption.S. Biomass Energy Policy. Earth Policy Institute.A. www. 2006. 2006. C. Energy Information Administration. EC 2008. Ethanol’s boom stalling as Glut depresses price. Available at: http://www.html. with sustainability doubts and financing Press Release. http://ec. Steenblik.consilium.managenergy.

Hazell and J. IEA (2006c) Energy Technology and Perspectives 2006 – Scenarios & Strategies to 2050. and allocation with a product performance or environmental quality standard: Illustration for the gasoline and additives market. International Energy Agency Paris. H. http://www. Renewable Fuels Agency. 14 December. World Energy. Paris OECD. Polasky.S. www. 30. P. International Journal of Production Economics. Gallagher.The Global Demand for Biofuels: Technologies. 2006. Price. Agricultural production and trade statistics and food balances. September 2008.ifpri. Gallagher.C. Ezzati and D. Gielen and F.K. Pachauri. Gallagher. Ezeji and H. Markets and Policies 51 T. A. IEA/ETO Working Paper. International Food Policy Research Institute – IFPRI Forum. economic. and P. Gallagher (2008) The Gallagher Review of the indirect effects of biofuels production. Biofuels. IEA (2006a) World Energy Outlook 2006. INFORMA.C. Polasky. Hazell and R. Biofuels: a win-win approach that can serve the poor.html. J.W. T. Schamel (2006b) The international competitiveness of the U. FAO.D. D.. C. http://www. H.htm. Who profits from the corn ethanol boom?. H. Harwood C. Sperling. 109 (5). D. 103–118. A low-carbon fuel standard for California. Kammen (2001) Quantifying the effects of exposure to indoor air pollution from biomass combustion on acute respiratory infections in developing countries. 29 May Focus Science. Inc.S. corn-ethanol industry: a comparison with sugar-ethanol processing in Brazil. 2005. National Renewable Energy Laboratory. Bioenergy: Microbial Contributions to Alternative Fuels. 103.W. Fargione.htm#focus. July. P.W. Journal of Policy Modeling.W. Food and Agriculture Organization. World biofuel demand to increase 20% annually.P. G. Proceedings of the National Academy of Sciences of the United States of America. International Food Policy Research Institute (IFPRI). Shapouri and G. International Energy Agency. 585–608. Faaij (2006) Outlook for advanced biofuels. Environmental Health 2 April. P. in Participation with MBI International and The Windmill Group. Energy Policy. Developed by Informa Economics. March 2006. and H. Shapouri. Paris http://www. 25. Hawthorne (2008) Land clearing and the biofuel carbon debt. http://www. risks and opportunities. Bioenergy and agriculture: promises and challenges – Overview. S. Gallagher (2007) A market and policy interpretation of recent developments in the world ethanol industry. and D. Biofuels: prospects. 230–245.N. Berkeley. Foust (2006) A research and market pathway to realize the potential of ethanol. UN Food and Agriculture Organization.php?nid¼377. Ag Decision Maker. Tiffany (2006) Environmental. Alternative Fuels – an Energy Technology Perspective. Farrell and pubs/catalog. Brubaker (2003) Some long run effects of growing markets and renewable fuel standards on additives markets and the U. Paris. J. Demain A. Bioproducts and Biorefining. J. http://aiche. P76222. Golden. pricing. International Energy Agency. P. Agribusiness. E. 481–488. Blaschek (2008) Practical aspects of butanol production. extension. 1235–1238. ethanol industry. P. S. M. D.P. 34 322–342. J. Hill.M. A. Hill. . E.iea. 101. Hamelinck and A. worldenergy.htm#focus. Shapouri and J. Freedonia.. and energetic costs and benefits of biodiesel and ethanol biofuels.S.confex. 22 (1). Faaij (2006) Bio-energy in Europe: changing technology choices. Tilman. Price (2006a) Welfare maximization. 34. Unander. FAOSTAT (2009) United Nations. Hofstrand. D. http://www.E. in: Wall J. IEA (2006b) IEA Statistics.iastate. The Emerging Biobased Economy: A multi-client study assessing the opportunities and potential of the emerging biobased economy. American Society for Microbiology Press. 3268–3283. The State of Food and Agriculture 2008. Schamel. Part 1: technical analysis. Energy Policy.iea.ifpri. 1(2). 2008. 109–134. (eds).org/pubs/catalog. 319 (5867). von Braun. Tilman.

2007. REN21. www. and J. 41 (23). 11 (1) January/February 2008. and R&D Needs.worldwatch. National Highway Traffic Safety Administration. G. RFA (2006a) From Niche to Nation – Ethanol Industry Outlook 2006.biofuelsdigest. NAE. Wright. Peppley (2006) Biomass for fuel cells: a technical and economic assessment.S. 2003.ppt. Fr€hlich. 12687. 20 November. . Ministry of Economy. Cambridge University Press. www.A. Lane. 28 December 2007.html. Assessment of Greenhouse Gas Emissions in the Production and Use of Fuel Ethanol in Brazil. http://www. Strategic Analysis of Opportunities for Brazil and the Hemisphere. Turhollow.html.D. a E. Perlack. NHTSA. July 2008.F. D. Japan seeks $1. Renewable Fuels Association. Biomass and Bioenergy. and node/5491. Berlin-Heidelberg.pdf. 32 (6). Reinhardt. March 2008. Europe May Ban Imports of Some Biofuel Crops. Watson. 201–218. Summary of Fuel Economy Performance. 2009. NYT. The Hydrogen Economy: Opportunities. Iowa State University. 50 pp. Graham.greencarcongress. 16. Intergovernmental Panel on Climate Change. National Academy of Engineering. Climate Change 2001: The Scientific Basis. Dhungana. 2008. 2004. ORNL/TM-2006/66. 2009. 2006. Nippon Oil To Mass-Produce ETBE in Japan. 7967–7973. Silva.php?nid¼109. 3. I.37 per gallon ethanol by 2015. R.agwatershed. Committee on Alternatives and Strategies for Future Hydrogen Production and nippon-oil-to-m. Biomass as Feedstock for Bioenergy and Bioproducts Industry: The Technical Feasibility of a Billion-Ton Annual Supply. M. USDA-DOE. Leal. Powering China’s development: The role of renewable energy. full report available at http://www.L. Determining the Regional Economic Values of Ethanol Production in Iowa Considering Different Levels of Local Investment. M. N.A. Energiegewinnung aus Biomasse. Externes o Gutachten. The National Mission on Jatropha Biodiesel. 28 April 2008. Barriers. L. http://www. A. 2007. Rainforest for Biodiesel? Ecological effects of using palm oil as a source of energy. November 2007. Trade and Industry. http://www. Costs.L. worldenergy. Economic Assessment of Biofuel Support Policies. Johnston and T. Renewables – Global Status Report – 2006 Update. B. DC. Energy Policy. Secretariat of the Environment – State of S~o Paulo. Japan’s Approach on Bioethanol. Prepared by Garten Rothkopf. 15 January. 35. access on Nill.52 Structure of the Bioenergy Business Inter-American Development Bank. Khanna. A Blueprint for Green Energy in the Americas. M. 2004. biodiesel production plunges to 22 percent of capacity. 2007. Environmental Science & Technology. excerpts from the Worldwatch special report: Powering China’s Development: The Role of Renewable Energy (Washington. mdic. Mathews (2007) Viewpoint biofuels: what a biopact between North and South could achieve. Gaertner. 2001. 2008. Renewable Energy World www. John and A. Paris: OECD Directorate for Trade and Agriculture. IPCC. N. International Journal of Green Energy. Jatropha World. and S.nhtsa. New York Times. S. accessed 22 January. Kaltschmitt.ren21. and R. http://ideas. and M.). Clifton-Brown (2008) Costs of producing miscanthus and switchgrass for bioenergy in Illinois.C. April 2007. The Agricultural Watershed Institute. World Wildlife Fund. Establishing a Grass Energy Crop Market in the Decatur Area: Report of the Upper Sangamon Watershed Farm Power Featuring: The global Biofuels Outlook. 3550– 482–493. OECD. Biofuels Digest. Holloway (2007) A global comparison of national biodiesel production potentials. World Energy. J. J. National Academies Press. Martinot and Li Junfeng. IFEU Institute. Macedo. Merten. B. Renewable Energy Policy Network for the 21st Century. Rettenmaier. U.

2004. Soyez (eds). J. Office of Energy Policy & New Uses. Erich-SchmidtVerlag. Houghton.iastate.E. The Netherlands: Copernicus Institute. D.htm. Wisner and P. S. Progress in Energy and Combustion Science. Scheffran and T. Searchinger. Economic Research Service. J.usda.worldwatch. 4 June. 160–185. USDA. and I. Estimating the net energy balance of corn ethanol. 1995. C. Markets and Policies 53 RFA (2006b) The Enhanced Small Ethanol Producer Tax Credit. 40(b)(3). Available at www. A Quickscan of Global Bioenergy Potentials to 2050: Analysis of the Regional Availability of Biomass Resources for Export in Relation to Underlying Factors. K. and K. 4– Dong. AgDM newsletter. U. 2008. Use of U. Yu. IEA Bioenergy. BioScience.C. Traynor. Tyner (2008) The US Ethanol and Biofuels Boom: Its Origins. Fabiosa. DC.The Global Demand for Biofuels: Briefing/Sugar/sugarpdf/EthanolDemandSSS249. and M.S. . September 2008. I. E. 2609–2614. Renewable Fuels Association. Science Express. pp. J. Bendor (2009) Bioenergy and Land Use – A Spatial-Agent Dynamic Model of Energy Crop Production in Illinois. Battaglini. Walter. Utrecht University. J. Office of the Chief Economist.S. Scheffran. Heimlich. 2006.A. Renewable Fuels Association. and Future Prospects. Study commissioned by AEATechnology as part of the Gallagher Biofuels Review for Renewable Fuels Agency. I. A. Graboski.pdf. Smeets. SSS-249. United Nations Environment Programme. R. Current Status. septc/documents/SEPTCPublication0601.A. 34. Baumel. 646–653.N. Turkenburg (2007) A bottom-up assessment and review of global bio-energy potentials to 2050. Duffield. M. E. J. P. in B. J.ethanolrfa. Green Jobs: Towards decent work in a sustainable. Rosillo-Calle and A. Romm (2006) The car and fuel of the future. United Nations. 2006. 2007. Smeets. www. The United Nations Biofuels Initiative. 56–106. The Promises and Challenges of Biofuels for the Poor in Developing Countries. Lewandowski. assessing indirect effects of biofuels on landuse change. Energy Policy. 19 April. www.Brennstoff a der Zukunft?. www. 2008. DC.S.pdf. August 2004. Tokgoz. U. Washington.ers. Energy for Sustainable Development. 7 February. Department of Agriculture. Critique of Searchinger (2008). Scheffran.pdf.ethanolrfa. EU set to scrap biofuels target amid fears of food crisis.B. 2006. Piacente. Butanol. Salassi.eurekalert. The Economic Feasibility of Ethanol Production from Sugar in the United States. Annual Report 2005–2006. Walter (2006) A global market for bioethanol: historical trends and future prospects. Worldwatch Institute: Renewables 2005: Global Status Report. A.pdf. Department for Transport. Will there be enough corn to supply future needs?.org/images/release_graphics/pdf/08 JulyAug Tyner.doc. Valdes. A. 39 (1/2). statistics. Faaij.S. Johnke. Lewandowski. and W. International Journal of Environment and Pollution. Borges da Cunha. Louisiana State University. and T. E. USDA. 12-6-2008. Hayes. Sylvester-Bradley. Berlin.R. Faaij.S. www. Market Evaluation: Fuel Ethanol. Pachauri. Shapouri. B.S. February. Elobeid.wikipedia. Energie und Klima. F. International Food Policy Research Institute. F.usda. Department of Agriculture/Baton Rouge. Ethanol Demand Driving the Expansion of Brazil’s Sugar Industry. 33. www. Weber (2004) Energie aus Biomasse und Bioabf€llen . http://www. 58 (7). Washington. An Analysis of the Effects of an Expansion in Biofuel Demand on U. Fairbanks. UN Foundation. Wiki. Dolzan. X (1). 2005. Department of Agriculture. U. croplands for biofuels increased greenhouse gases through land-use W. 2007. R. and M. Agriculture. 2008. January 2007. I. RFA (2008) Industry Statistics. H. www. and J. J. A.bioenergytrade. Sec. 18–30.K. Wikipedia 2008. R. Agricultural Economic Report Number 721. http://www. F. unfoundation. http://en. low-carbon world. von Braun and R. Utrecht. ADAS UK Ltd. T. Sugar and Sweeteners Outlook.extension. UNEP. The Guardian.

Washington State Department of Agriculture. van de Vooren (eds). . Available at http://agr. 2008. WSDA. London: Earthscan. 2007. 7 March. Global Potential and Implications for Sustainable Agriculture and Energy. NM: International Relations Center. http://americas.html. Silver City.irc-online. Bioenergy: frequent asked questions. R. Sugarcane ethanol . Wageningen Academic Publishers. P.wa. Zuurbier. J.Contributions to climate change mitigation and the environment. Zibechi. Biofuels for Transport.54 Structure of the Bioenergy Business Worldwatch Institute. United States and Brazil: The New Ethanol Alliance.

which supply about 80 % of the world’s energy. Hughes and Nasib Qureshi 3. 2005). 9 %) is traditional biomass used mainly in inefficient ways. sugar. defined as all plant and plant-derived materials. in addition.3 Biofuel Demand Realization Stephen R. of which the largest fraction (ca. and has fewer environmental concerns than fossil fuels. animal manure. inexpensive. including forestry residues. It is estimated that forest and agricultural lands alone – the two largest potential biomass sources – could produce enough biofuels to meet more than one-third of the current demand for transportation fuels (Perlack et al. renewable energy represents only about 14 % of the total world energy supply. Nasib Qureshi. waste from pulp and paper mills. which is in the public domain in the United States of America. and oil crops already used for energy.. However. are projected to be depleted within one or two generations at the present rate of consumption and. . it is more labor-intensive and the costs of transportation and processing are still a barrier to widespread use. Biomass. and urban wood waste. not just starch. is projected to be a growing part of future sustainable energy sources. At the present time. Fossil fuels. Biomass is widely available.1 Introduction Achieving a sustainable energy future depends increasingly on renewable energy sources. Hans Blaschek e and Hideaki Yukawa The contribution of Hughes and Qureshi has been written in the course of their official duties as US government employees and is classified as a US Government Work. 2007). Nontraditional biomass (biomass used in a sustainable way) currently provides about 2 % of the total energy consumption in the US. have environmental and security concerns (Goldemberg. such as wood burned for cooking in rural areas. Biomass to Biofuels: Strategies for Global Industries Edited by Alain Verts. The full resource potential might be realized in several decades when large-scale bioenergy industries and combined biorefineries are likely to exist.

from 2006 to 2007. driven by EU demand (FAPRI. brought about by EISA (FAPRI. countries are promoting ethanol and biodiesel for use as a biofuels with bioenergy mandates and directives.70 per gallon. coupled with a weak dollar.1 Availability of Renewable Resources to Realize Biofuel Demand Demand for Ethanol and Biodiesel Throughout the world. a coproduct in sugar production from sugarcane.2 BG in 2007. is projected to drive the world price to about US$ 5 in 2008.2. an increase from 2006 of about 15 %. have all contributed to sharp increases in US grain and oilseed prices.0 BG. was 430 million gallons. With the decrease in US imports in 2007 and the increase in ethanol supply. was 5. consumption was 215 million gallons. The US. Brazil’s ethanol production using sugar cane totaled 5.2. and because of high crude oil prices. as production continues to increase this downward trend will continue. production being projected to reach 2. 2009). The increasing demand for biodiesel as EU countries attempt to achieve their biofuel targets. consumption is projected to reach 217 million gallons and production 468 million gallons by 2017. the mandates of Argentina and Brazil for 5 % biodiesel blend by 2010. the world economy and the weather. Among these energy policies are the 2003 Renewable Fuels Directive of the EU. 2009).56 Structure of the Bioenergy Business 3. In the EU. 2009).2 % over 2006. Although.2 Status of First-Generation Biofuel Feedstocks The expanding demand for biofuels. 2009). by 2017. In China. ethanol production decreased by 2.4 BG in 2006 and approximately 7. and producer incentives in Canada.2 BG in 2007. produced 6. the price declined by 12.5 BG of ethanol in 2007 compared to 4. Chinese fuel ethanol production. with China and India emerging as significant producers. 3. an increase of almost 15. this will require 24 billion gallons (BG) of biofuels by 2017. ethanol production in India was 594 million gallons in 2007 (FAPRI. it is expected to be reversed by 2012 because of a higher ethanol demand from the US. the 2007 Energy Independence and Security Act (EISA. Brazil’s biodiesel production is driven by its mandate and the international market. 2009).0 BG in 2007 (RFA. by using primarily corn as the feedstock.2 3.5 BG and consumption expected to reach 3. 2009). Similar to Argentina. and in India it was 480 million gallons in 2007. In India.9 BG in 2006 (Renewable Fuels Association. Fuel ethanol consumption in Brazil was 4. the world price is projected to increase again to about US$ 6 by 2017. biodiesel use is projected to reach 634 million gallons by 2017. US fuel ethanol demand. using primarily corn. including production and imports.2 % to 570 million gallons because of higher feedstock prices. Although an expanded production in Argentina and Brazil will lead to a temporary price decline in 2009. Consumption in the EU reached 1. an increase of less than 1 % over 2006. and production to increase to 718 million gallons (FAPRI. Brazil and the United States are the major producers of fuel ethanol. The EU has the world’s most developed biodiesel industry with.6 % to $1. fuel ethanol is produced mainly from molasses. In the US.3 BG in 2007. New energy legislation and high petroleum prices are expected to contribute to a continued strong . In Argentina. 2007) mandates minimum levels of domestic use of classes of biofuels.

Taiwan. South Africa) are projected to increase their production to meet rising world demand.2 million tons with reductions for China. The largest increase in demand for corn comes from Asian countries because of growth in their livestock industries and thus a demand for feed. Despite some late December 2008 dryness that delayed plantings in western growing areas. The USDA projected a 40 million-bushel increase in sorghum use in early 2009. The expansion in ethanol production has caused the amount of corn used for fuel ethanol to exceed US corn exports. Brazil.4 million tons on lower yields (USDA WASDE.5 million ton increase for Kenya imports. South African production is projected to be 1. 2009). and Mexico (USDA WASDE. and thus boost supply. The early season drought that continued in southern growing areas through February 2009 . Global corn demand had increased in 2008. but partly offsetting this reduction is a 0. 2009). 2009). with reductions in expected feed. seed.0 million tons higher. Corn imports for 2009 are lowered by 0.0 million tons higher. World corn production for 2009 is projected to increase by 0.5 million tons higher in 2009 than in 2008. and industrial use is also lowered 2.6 million tons.0 million tons.3 million tons with a lower projected harvested area and yields.0 million ton reduction for China and a 0. Global corn consumption is lowered. Corn exports are projected to be lower by 50 million bushels due to increased foreign supplies of corn and wheat (USDA WASDE. and industrial uses. as higher ethanol use would more than offset a reduction in exports. and then to remain relatively stable. Increases are also projected for South Africa. 2009). particularly in the US. the main US competitors in the corn market (Argentina. Rainfall was above average in both January and February 2009. Average corn prices in 2007 – 2008 have approximately doubled over what they were two years ago (FAPRI. Mexico will increase its imports of US dried distillers grains in 2009 (FAPRI. Global sorghum production. however.Biofuel Demand Realization 57 growth in biofuel production. weather conditions throughout the Maize Triangle were extremely favorable in late 2008. As a consequence. South Africa and the Philippines. seed. Rising corn prices resulted in an increase in corn acreage of 15 million acres in 2007. Global corn supplies for 2009 are projected to be 8. and South Africa. In early 2009. increasing the world price by more than 27 % to about US$ 200 per metric tonne. with an increase for India unable to offset a projected decrease in Argentinian sorghum production of 1. based on indications of increased sorghum use by ethanol plants in the US Southern and Central Plains. Malaysia.6 million ton reduction for India. and in Kenya of 0. Feed use is lowered 2. at 12. is projected to be 0. Indeed. 2009). In the next decade.5 million tons on lower harvested area and yields. mainly as the result of an increase for South Africa. Food. This will be offset by an expected reduction in 2009 corn production in India of 0.5 million tons each for Malaysia and Taiwan. Argentina. Government procurement policies in China are expected to reduce industrial corn use. with the largest increase for China where amounts are projected to be 6.5 million tons lower in 2009. Use of corn as a feedstock for ethanol in the US is projected to be 100 million bushels higher in 2009 because of improving incentives for gasoline blending and greater ethanol use. US corn supplies were projected to be 50 million bushels lower for the coming year. more than offsetting increases for the US.2 million tons with a 5. food. Blender margins have become increasingly favorable as gasoline prices have increased relative to those for ethanol. Lower expected corn exports for the US are only partly offset by small increases for India and Russia. including ethanol. the price is expected to decrease slightly in 2009 as production increases.

Whilst most of these are currently being used. corn stover (stalks. forest lands have the potential to sustainably produce close to 370 million dry tons of biomass annually.1 million tons due to drought in South America (USDA WASDE. process . The US soybean acreage is expected to rebound in 2008.1 million tons to 224. husks). urban waste (paper. which adds significantly to the cost of the final product.2. although supplies will remain short and the prices high (FAPRI. and agricultural residues such as sugar cane waste (stalks. and 106 million dry tons of animal manure. Cellulosic biomass is viewed as a promising source for providing renewable energy for the future. stem tips). and 60 million dry tons from fuel treatment operations. 47 million dry tons of urban wood residues (including construction debris). 2005). 2009). woody plants). wheat straw. Sorghum production for India is projected to be 0. Global efforts are underway to develop cost-effective conversion technologies to create opportunities for cellulosic waste to become a major biofuel feedstock. tree trimmings. as mandated by EISA in the US. Acreage shifts caused a sharp reduction in 2007 soybean production. 2005). and rice straw (Kojima and Johnson. 2009). the demand for US soybeans is expected to remain strong because of income-driven demand growth in China and the remainder of Asia. cobs. 2009). 2009).58 Structure of the Bioenergy Business increased the expected abandonment and reduced yield prospects for this year’s crop. Projected US soybean supplies for 2009 were reduced to 210 million bushels. The estimated global soybean production in 2009 was reduced by 9. forest thinnings. leaves. and transported to conversion facilities. the overall level of advanced or second-generation biofuels – that is.3 Status of Second-Generation Biofuel Feedstocks Production from first-generation biofuel feedstocks alone will not be able to satisfy the world’s growing energy needs.7 million tons on higher reported yields (USDA WASDE. 2009). This estimate includes 428 million dry tons of crop residues. and supplies are plentiful with this year’s slower export pace (FAPRI. 87 million dry tons of grains used for biofuels.. 145 million dry tons of residues from wood processing mills and pulp and paper mills. and because of the global demand for vegetable oil to make biodiesel. However. grass clippings). These materials must be collected. and still continue to meet food. biodiesel. This projection includes 52 million dry tons of fuelwood harvested from forests. The US 2009 season-end price projection is about US$ 9 per bushel. processed. and urban wood residues. Agricultural lands in the US can provide close to 1 billion dry tons of collectable biomass. and export demands. The world sorghum prices are well below those for corn in these areas. 377 million dry tons of perennial crops. The biomass resource base is composed of a wide variety of forestry and agricultural resources. two potentially large sources of forest biomass not currently used are logging residues and fuel treatment thinnings. feed. industrial processing residues. These could include: logging residues. In the US. Of the total requirement of 24 BG of biofuels by 2017. 64 million dry tons from logging and site-clearing operations. which are projected to contribute more than 120 million dry tons annually (Perlack et al. cellulosic ethanol and other biofuels not made from corn – will need to increase from 600 million gallons in 2009 to 9 BG by 2017 (FAPRI. wood mill residues. 3. leaves. energy crops (switchgrass. contributing to decreased supplies and much higher soybean prices.

2005) The European Union (EU) will need to produce an estimated 11. Bagasse.. China. producing 253 million liters. An ability of microbes to tolerate any inhibitory byproducts produced during lignocellulose pretreatment. . Colombia.. while 580 million liters was derived from wheat. 2007). which accounted for 976 liters. with a total production of 430 million liters mainly from rye. 2008).. A study prepared for the US Department of Energy assessed the availability of cellulosic feedstocks in Argentina. More efficient pretreatment processes that open the structure of biomass (and/or generate fractionated component streams) with reduced production of inhibitory byproducts.Biofuel Demand Realization 59 residues. . and other residues generated in food production. Canada. the EU produced close to 1. Consolidated bioprocessing microbes that can produce their own lignocellulosedeconstructing enzymes (some or all) and ferment all sugars to ethanol. . . This estimate includes 187 mmt from bagasse. An ability of microbes to tolerate industrial conditions (robustness to change) and also to tolerate high ethanol concentrations.6 billion liters of fuel ethanol primarily from cereal grains. . Potential supply projections of cellulosic feedstocks in these countries from forestry activities amounted to 154 mmt. and include grasses and woody plants. and Mexico. The projected amount of recoverable crop residues for 2017 in those countries was about 246 million metric dry tonnes (mmt). In 2006. simultaneously. India. and 2 mmt from palm oil processing wastes (Kline et al. The perennial crops are crops dedicated to bioenergy and biobased products. the crushed stalk residue from sugar cane processing. Of the total 488 mmt total cellulosic supply projection.6 billion liters of bioethanol to meet its 2010 target of a 5. is by far the most important single cellulosic resource in the countries studied.75 % share of energy derived from renewable sources. 17 mmt from wheat straw. An ability to conduct simultaneous saccharification and fermentation (SSF) at elevated temperatures (>50  C). The provision of this level of biomass can be accomplished with relatively minor changes in land use and agricultural practices (Perlack et al. Brazil. An ability to deal with the problem of low bulk density of the feedstock which limits ethanol concentration: . about half is projected to be valued at US$ 36 per dry tonne or less in 2017 (Kline et al. More importantly.1 Technology Improvements to Enhance Biofuel Production Economics Technology Overview To achieve the goal of integrated biorefineries that can use a broad range of feedstocks. followed by wheat (Biofuels International. so as to maximize the conversion rate and minimize contamination. Supplies from perennials were estimated to range from 50 to 100 mmt. 3. the following technology improvements are necessary for profitability: .3 3.3. bagasse is conveniently available at sugar cane ethanol refineries and is estimated to be the most economic cellulosic resource. Sugarbeet serves as another ethanol feedstock. 2008). 40 mmt from corn stover. The largest biofuel producer in 2006 was Germany.

and to high ethanol concentrations. switchgrass. Genes encoding lignocellulose-deconstructing enzymes could also be inserted into the yeast strain engineered for xylose utilization.1) for use in cellulosic ethanol production. 2008. Stable yeast strains were engineered using these gene sets to allow fermentation of cellulosic hydrolysates that contain arabinose and xylose. to the presence of inhibitors. Strategies for producing key improvements in the microbes used for the production of fuel ethanol include: 1. tolerance to inhibitors. A stable industrial cell line will be established to produce a highly durable background strain (GMAX. Currently. . characteristics such as activity at elevated temperatures. which are the principal constituents of hard and soft woods. such as engineering microbes to express enzymes capable of deconstructing lignocelluloses (Kumar et al. Recombinant yeast strains have been engineered that utilize glucose and xylose. 2006). Saccharomyces cerevisiae is a robust organism with high tolerance to inhibitors such as furfural and methyl furfural. and robustness have been considered more difficult to impart. 2005) or expanding their ability to utilize all the sugars in biomass hydrolysates (Van Maris et al. using glucose as a substrate.. Although ethanol production from xylose is limited. most of this effort has focused on traits that are thought to be more amenable to manipulation. a comparison of ethanologens is under way to determine the organism best suited for further development for fuel ethanol production (Hahn-H€gerdal et al. which yields a maximum of 6 % ethanol on glucose with limited oxygenation. yeast have several distinct advantages over bacterial ethanologens. utilizing a membrane-based system to recover ethanol by pervaporation integrated with fermentation. corn stover. The resultant yeast strain would be selected for its tolerance to processing temperatures. This is in addition to glucose fructose. . the xylose could be used for cell growth. Figure 3. is presented in Table 3. but do not ferment xylose to ethanol in high yield. To date. or which are under consideration for use as ethanologens.submerged SSF. . The tolerance of S. 2007). For example. Yeast strains that were systematically transformed with yeast libraries were screened to identify key genes that could regulate xylose and arabinose utilization.1 (Kurtzman. Yeast are also far better at tolerating a low pH than are many bacteria. Lynd et al. . or . and corn cobs. mannose and galactose. .3.2 Engineering Improved Bioprocessing Microbes Considerable research has been conducted to improve the characteristics of bioprocessing microbes.60 Structure of the Bioenergy Business . Engineering one microbe with the ability to ferment mixed sugars: . cerevisiae to 18 – 20 % ethanol..solid-state SSF. is significantly higher than that of the yeast Pichia stipitis (now named Schefferomyces stipitis). as well as to the ethanol produced.. 3. A comparison of microbes that are currently used. utilizing novel reactor designs with ethanol recovery via steam stripping. At this a point. 2009). On the other hand.. allowing all of the glucose to be used solely for fermentation to ethanol.

galactose. arabinose. arabinose Glucose. sorbose. fructose. sucrose. mannose (continued) . rhamnose. xylose. mannose 61 Glucose. xylose. glucuronic acid. galacturonic acid. sucrose. maltose. glucuronic acid. sucrose. fructose. sucrose. arabinose. xylose. mellibiose. fructose. mannose Glucose. xylose. arabinose Glucose. xylose. maltose. isomaltose. sucrose. mannose Candida shehatae or Pachysolen tannophilus Kluyveromyces marxianus Escherichia coli (FBR2) Zymomonas mobilis (Zm4) Ethanologen trait Saccharomyces cerevisiae Sugars metabolized Glucose. maltotriose Glucose. galactose. galactose. maltose. isomaltose. fructose.1 Microbes currently used or under consideration for use as ethanologens Scheffersomyces stipitis (formerly Pichia stipitis) Glucose. galactose. fructose. raffinose. galactose. isomaltose. maltotriose. arabinose. xylose. raffinose. galactose. maltose. trehalose. arabinose Glucose. raffinose. arabinose. cellobiose. maltose. xylose. sucrose. maltotriose. maltose. trehalose. fructose. sucrose. arabinose. arabinose. isomaltose. fructose. galactose. fructose. maltotriose. sucrose. lactose. isomaltose. mellibiose. lactose Glucose. trehalose. xylose. sucrose. trehalose. raffinose. isomaltose. galactose. xylose. raffinose. galactose. maltose. arabinose Glucose. trehalose. maltose. lactose. sucrose. fucose. sucrose. fructose. raffinose. sucrose. fructose. xylose. glucuronic acid. galactose. maltose.Table 3. fructose. raffinose. xylose. maltotriose. maltotriose. and have been engineered to use lactose. galactose. maltose. raffinose. and maltotetrose Glucose. lactose. maltose. arabinose Biofuel Demand Realization Sugars fermented Glucose. maltose. raffinose. fructose. trehalose. ribose. ribose.

0–7.8 8.4–6.5  C 5.8 % (<2 h) Crabtree-negative (glucose taken up by proton symport) Neither strain fully sequenced Not fully sequenced Crabtreepositive.3 4.5–3.6–4.8 %= <4.0–8.1–10.3 6. sequence maintained at MIPS Comprehensive Yeast Genome Database (CYGD) DOE JGI data Sugars to pyruvate using engineered Entner– Doudoroff pathway The Genome Center at the University of Wisconsin maintains sequence .5 4.0 15–21 %= <22–23 % Structure of the Bioenergy Business Temperature for growth pH range Ethanol production/ tolerance Crabtree type and/or metabolic flux 3.1 (Continued) Scheffersomyces stipitis (formerly Pichia stipitis) 26–35  C 4.0–11.5 %= <15 % Sugars to pyruvate using the Entner– Doudoroff pathway DOE JGI data sequence not complete 3.5 3.1 %/ <22.38 %/<5 % 10–40  C <40  C <49  C Candida shehatae or Pachysolen tannophilus Kluyveromyces marxianus Escherichia coli (FBR2) Zymomonas mobilis (Zm4) Ethanologen trait Saccharomyces cerevisiae <44  C 27–37.0–7.8–6.5–6.8–6.0 %= <10 % Crabtreenegative Crabtreenegative 4.62 Table 3.5 % 4. Genome sequence sequence completed uses facilitated diffusion of glucose Max Planck Institute for Biochemistry Martinsried/ Munich.

. . or in the wet mill process to utilize pericarps for cellulosic ethanol production. or with lignol moieties from lignin to produce bioderived plastics. such as lipases for transesterification or hydrolases for saccharification. . Many low-cost enzymes.1 Schematic of the recombinant GMAX yeast strain This same strain also allows the production of ethanol from sugar cane. The fermentation of glycerol and thin stillage obtained from the ethanol production process to 2-methyl-1. sugar beet and sweet sorghum.3-propanediol and 1. The propanediol moieties can be used for condensation polymers with terephthalic acid. respectively. have been shown to be functional in yeast (Den Haan et al.. 2.3 propanediol will require the additional microbes Citrobacter freundii and Clostridium butyricum. This flexibility will be needed to allow the use of dedicated energy crops in biorefineries where ethanol is produced from both lignocellulose and sucrose. so as to produce ethyl esters for biodiesel. Starch operations could use this same yeast not only for ethanol production but also to process corn stover.Biofuel Demand Realization 63 Figure 3. . 2007). Yeast strains can also be engineered to express lipase enzymes to catalyze the ethanol transesterification of triacylglycerides from corn oil. . Engineering the microbe to produce multiple products or using additional microbes: . The GMAX yeast strain can provide an expression setting to produce low-cost enzymes for production processes in a combined biorefinery. as for sugar cane with bagasse and sucrose or for sugar beets with high concentrations of sucrose and cellulosic waste.

. 3. Selected optimized genes or gene sets will be added to the improved GMAX strain. depending primarily on the use of the product. Adding desired metabolic capabilities. The resultant strains would be screened for survival on medium containing inhibitors or high amounts of ethanol. Additional selected genes will be added to the GMAX strain if evaluation demonstrates that the fermentative pathways in S.4. and the activity of the resultant strain evaluated after each addition. The treatment strength would determine how many multigene changes are produced.1 Before any commercialization of the products of recombinant organisms can be realized. The metabolic pathway involving these enzymes directs some xylose into the production of this sweetener so as to make the highnutrient cattle feed more palatable. zein can be expressed as homozein types with desired polymer properties. . 4. using routes such as mutagenesis or the addition of multiple FLEXGene collections. The product can be added as a high-value nutritional feed supplement and pelletized for shipment. and incubated anaerobically at high temperatures. A second approach would be to take the stable GMAX yeast strain and combine the four-plasmid expression with multiple fungal or bacterial expression libraries simultaneously for multigene interactions. certain regulatory requirements must be met. such that the use of glucose remains intact and the genes for pentose utilization still provide the capacity for the strain to metabolize xylose and arabinose. Existing refineries can use these lignocellulosic add-ons to generate high – value coproducts and use low-revenue feed items from the process for higher-value plastics and food-grade polymers. . Further enhancements of the GMAX strains will require multigene additions or changes. For example. cerevisiae are not disrupted. The nutrient value of animal feed can also be improved by using the yeast to express proteins having high proportions of essential amino acids. The regulation of products of modern biotechnology in the US and elsewhere is complex . . followed by selection to develop tolerance to ethanol and inhibitors: . . One method of accomplishing such changes would require the mutagenesis of the strain by treatment with chemicals or radiation. Both routes would require ultra high-throughput screening.64 .4 US Regulatory Requirements for Organisms Engineered to Meet Biofuel Demand Challenges of Regulating Biotechnology 3. Various routes of producing xylitol are possible with yeast expressing xylose isomerase and xylitol dehydrogenase. as the factorial combinations of genes involved in this approach is enormous. Structure of the Bioenergy Business The expression of saccharification enzymes in the GMAX yeast would drastically reduce any requirement for the addition of enzymes in cellulosic ethanol production. Determining the most logical sequence to add the desired traits: . 3. metabolic adjustments will be required to maintain industrial doubling times and superior ethanol production. Further metabolic engineering is necessary after addition of genes for any of the enzymes as these will inherently impact cell growth.

Biofuel Demand Realization


and still evolving, although in the US the key federal agencies have remained the same for some time. These include the Food and Drug Administration (FDA), the Environmental Protection Agency (EPA), and the Animal Plant Health Inspection Service (APHIS) within the US Department of Agriculture (USDA). Since the first commercial applications of biotechnology during the early 1980s, almost three decades have elapsed, and the product types, regulations and policies have changed considerably over that period. The technique of genetic engineering – in which recombinant DNA technology is used to introduce desirable traits into organisms – provides powerful tools to enhance animals, plants, and microbes for the benefit of society. The development of microbes such as yeast that are engineered to ferment the constituent sugars in lignocellulosic biomass are one such example, but the process may involve many regulatory agencies. Currently, microbes are being custom-engineered not only to ferment the sugars in such feedstocks but also to express high-value coproducts (Hughes et al., 2008; Hahn-H€gerdal et al., 2007). Genetic a engineering poses unique challenges to the regulatory process because of the wide variety of changes that can conceivably be created in almost any organism (Falk et al., 2002). State laws and local ordinances also can apply, especially in the area of any deliberate or planned releases of genetically altered organisms into the environment.


Biotechnology Regulatory Systems in the US

The systematic regulation of biotechnology by the federal government officially began on 23 June, 1976, when the National Institutes of Health issued Guidelines for Research Involving Recombinant DNA Molecules (NIH, 2002) to oversee research activities. These guidelines cover all aspects of recombinant DNA research, and they were – and still are – mandatory only for an institution that receives funds from the NIH; however, they are widely accepted and followed, even by institutions that do not receive NIH funding. The purpose of the NIH Guidelines is to specify practices for constructing and handling: (i) recombinant deoxyribonucleic acid (DNA) molecules; and (ii) organisms and viruses containing recombinant DNA molecules. In the context of the NIH Guidelines, recombinant DNA molecules are defined as either: (i) molecules that are constructed outside living cells by joining natural or synthetic DNA segments to DNA molecules that can replicate in a living cell; or (ii) molecules that result from the replication of those described in (i). Any recombinant DNA experiment which, according to NIH Guidelines, requires approval by the NIH, must be submitted to the NIH or to another federal agency that has jurisdiction for review and approval. Once approvals, or applicable clearances, are obtained from another federal agency the experiment may proceed without NIH review. The US Department of Agriculture, Animal and Plant Health Inspection Service (APHIS) is the federal agency that has regulatory control over the introduction, including importation, interstate movement, or environmental release, of genetically engineered organisms that may pose a plant pest risk, including plants, insects, or microbes. In August 2002, the USDA created a new unit within APHIS, called Biotechnology Regulatory Services (BRS), to focus on the USDA’s key role in regulating and facilitating biotechnology (FDA, 2002). BRS derives its authority from the Federal Plant Protection Act (PPA), which is a part of the Agriculture Risk Protection Act of 2000. According to the Federal


Structure of the Bioenergy Business

PPA, any transgenic crop containing DNA of a known plant pest is viewed as a potential plant pest. All regulated introductions of genetically engineered organisms must be authorized by APHIS under either its permitting or notification procedures. APHIS issues permits for the introduction of genetically engineered organisms that pose a plant pest risk, including plants, insects, or microbes. All regulated genetically engineered organisms are eligible for the permitting procedure (USDA APHIS, 2009). Applicants submit scientific information for APHIS to review before APHIS issues the permit. Notification is an administratively streamlined alternative to a permit (USDA APHIS, 2008). Developers must meet rigorous containment, safety, and performance criteria for APHIS to accept the notification. When the developer has collected sufficient data, APHIS will accept a petition to deregulate the crop. APHIS grants nonregulated status if the genetically engineered organism poses no more of a plant pest risk than an equivalent nonengineered organism. If a regulated article is very similar to a genetically engineered organism that has already been granted nonregulated status, then APHIS may extend nonregulated status to that organism. Nonregulated status means that permits and notifications are no longer required for the introductions of this organism (USDA APHIS, 2009). The regulations governing biotechnology, as overseen by APHIS BRS, are found in the Federal Register and in the Code of Federal Regulations, Title 7, Section 340 (7CFR 340). Rapid developments in genomics are resulting in dramatic changes in the way that new plant varieties are developed and commercialized. Scientific advances are expected to accelerate over the next decade, leading to the development and commercialization of a greater number and diversity of bioengineered crops. USDA/APHIS oversees the field testing of new varieties of bioengineered plants and requires developers to follow procedures that minimize the chance of inadvertent introduction of material from these new varieties to agriculture, the environment, and the food supply. As the number and diversity of field tests for bioengineered plants increase, however, the likelihood that crosspollination due to pollen drift from field tests to commercial fields and commingling of seeds produced under field tests with commercial seeds or grain may also increase. This could result in the inadvertent, intermittent, low-level presence in the food supply of proteins that have not been evaluated through FDA’s voluntary consultation process for foods derived from new plant varieties (referred to as a ‘biotechnology consultation’ in the case of bioengineered plants). In June 2006, the FDA (Center for Food Safety and Applied Nutrition/Office of Food Additive Safety and CVM) issued a Guidance for Industry, Recommendations for the Early Food Safety Evaluation of New Non-Pesticidal Proteins Produced by New Plant Varieties Intended for Food Use, to address this possibility (FDA CFSAN, 2006). This guidance describes the procedure for early food safety evaluation of new proteins in new plant varieties that are under development for food use. Since FDA first issued its 1992 policy (FDA, 1992 Policy), the agency has encouraged the developers of new plant varieties – including those varieties developed through biotechnology – to consult with the FDA early in the development process to discuss any possible scientific and regulatory issues that might arise. This current guidance continues to foster early communication by encouraging developers to submit to the FDA their evaluation of the food safety of their new protein. Such communication helps to ensure that any potential food safety issues regarding a new protein in a new plant variety are resolved early in development, prior to any inadvertent introduction into the food supply of material from that plant variety. The submission of an early food safety evaluation for a new

Biofuel Demand Realization


protein is not meant to substitute for a biotechnology consultation with the FDA about a food derived from a new bioengineered plant variety. If a developer decides to commercialize a new bioengineered plant variety, the FDA expects that the developer will participate in the consultation process (FDA CFSAN, 1997). The FDA concluded that it is sensible for developers of new varieties of foods to consult with the FDA so that “. . .relevant scientific, safety, and regulatory issues are resolved prior to the introduction of such products into the marketplace.” To facilitate this consultation process, the Biotechnology Evaluation Team (BET) was created by the FDA’s Center for Food Safety and Applied Nutrition (CFSAN), Office of Surveillance and Compliance (OSC), and Center for Veterinary Medicine (CVM). The BET is composed of a consumer safety officer, molecular biologist, chemist, environmental scientist, toxicologist, nutritionist, and may include supplemental expertise depending on the product in question. The BET requests information, such as the intended uses of a food, the source of genetic material, the intended technical effect of the modification, the expected effect on the composition of the food, the identity, function, and concentration of all newly expressed products, any known or suspected allergens or toxins, a comparison of the composition of the new food to that of the parental variety, and any other relevant information concerning the safety and nutritional assessment of the bioengineered food. Developing this information can require several years. In the Federal Register of January 18, 2001 (66 FR 4706), the FDA (FDA, 2001) issued a proposed rule that would require developers to submit a scientific and regulatory assessment of the bioengineered food 120 days before the bioengineered food is marketed. In this premarket notification proposal, the FDA recommends that developers continue the practice of consulting with the Agency before submitting the required premarket notice. One additional regulatory consideration pertains to the use of antibiotics in ethanol production facilities. The FDA does not have regulatory authority over the use of antibiotics in fuel ethanol plants. However, it does have authority over animal feeds, and thus over the use of distiller’s grains for animal feed and over any antibiotic residuals in these grains for animal feed. LactrolÒ antimicrobial (virginiamycin þ dextrose), was developed by SmithKline Beecham following meetings and correspondence between the FDA CVM in 1992 – 1994 about how to meet FDA requirements. In 1993, the FDA CVM provided a letter of ‘no objection’ to the developer for the use of a formulation of virginiamycin and lactose in ethanol fermentation. The formulation was later changed to virginiamycin and dextrose, with FDA/CVM’s assent.



It is anticipated that many of the problems associated with first-generation biofuels can be addressed by the production of second-generation biofuels manufactured from agricultural and forest residues, and from nonfood crop feedstocks. Where cellulosic feedstock is to be produced from dedicated energy crops grown on arable land, concerns remain over competing land use, although energy yields are likely to be higher than if crops grown for first-generation biofuels were produced on the same land. In addition, poorer quality land could possibly be utilized. Given the current investments being made to gain improvements in technology, it is expected that, in the long term, the second-generation


Structure of the Bioenergy Business

biofuels will reach full commercialization. In the near future it is likely that the infrastructure and experiences gained from the production of first-generation biofuels will be transferred to support second-generation biofuel development. The full potential of biofuel production will be realized in several decades, when large-scale bioenergy industries and combined biorefineries are likely to exist.

Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply reccommendation or endorsement by the United States Department of Agriculture.

Biofuels International. Feedstocks of the future. Biofuels International Newsletter Online September 25, 2007. R. Den Haan, S.H. Rose, L.R. Lynd, and W.H. Van Zyl. Hydrolysis and fermentation of amorphous cellulose by recombinant Saccharomyces cerevisiae. Metab. Eng., 9, 87–94 (2007). Energy Independence and Security Act (EISA) of 2007. H.R. 6, 110th US Congress, Washington, D.C. (2007). M.C. Falk, B.M. Chassy, S.K. Harlander, T.J.IV. Hoban, M.N. McGloughlin, and A.R. Akhlaghi. Food biotechnology: benefits and concerns. J. Nutr., 132, 1384–1390 (2002). Food and Agricultural Policy Research Institute. FAPRI 2008 US and World Agricultural Outlook. Department of Economics, Iowa State University (2009); outlook2008/. Food and Drug Administration, Center for Biologics Evaluation and Research, Center for Drug Evaluation and Research, Center for Veterinary Medicine, USDA, APHIS, CVB, BRS. Drugs, biologics, and medical devices derived from bioengineered plants for use in humans and animals; Draft guidance. September 2002. Food and Drug Administration Center for Food Safety and Applied Nutrition/OFAS CVM. Guidance for Industry, Recommendations for the Early Food Safety Evaluation of New Non-Pesticidal Proteins Produced by New Plant Varieties Intended for Food Use, June 2006. Food and Drug Administration. Statement of Policy: Foods derived from new plant varieties. Fed. Reg., 57 (104), 22984–23001 (1992). Food and Drug Administration Center for Food Safety and Applied Nutrition. Guidance on consultation procedures. Foods derived from new plant varieties (1997); 1rd/consulpr.html. Food and Drug Administration. Premarket notice concerning bioengineered foods. Fed. Reg., 66 (12), 4706–4738 (2001). J. Goldemberg, Ethanol for a sustainable energy future. Science, 315, 808–810 (2007). B. Hahn-H€gerdal, K. Karhumoa, C. Fonseca, I. Spencer-Martins, and M.F. Gorwa-Grauslund. a Towards industrial pentose-fermenting yeast strains. Appl. Microbiol. Biotechnol., 74, 937–953 (2007). S.R. Hughes, D.E. Sterner, K.M. Bischoff, R.E. Hector, P.F. Dowd, N. Qureshi, S. Bang, N. Grynaviski, T. Chakrabarty, E.T. Johnson, B.S. Dien, J.A. Mertens, R.J. Caughey, S. Liu, T. Butt, J. LaBaer, M.A. Cotta, and J.O. Rich. Three-plasmid SUMO yeast vector system for automated high-level functional expression of value-added co-products in a Saccharomyces cerevisiae strain engineered for xylose utilization. Plasmid, 61 (1), 22–38 (2008). K.L. Kline, G.A. Oladosu, A.K. Wolfe, R.D. Perlack, V.H. Dale, and M. McMahon.Biofuel feedstock assessment for selected countries (ORNL/TM-2007/224). Oak Ridge National

Biofuel Demand Realization


Laboratory, Oak Ridge, Tennessee, managed by UT-Battelle, LLC for the U.S. Department of Energy (2008). M. Kojima, and T. Johnson Potential for biofuels for transport in developing countries. The International Bank for Reconstruction and Development, The World Bank, Energy Sector Management Assistance Programme Report (2005). R. Kumar, S. Singh, and O.V. Singh. Bioconversion of lignocellulosic biomass: biochemical and molecular perspectives. J. Ind. Microbiol. Biotechnol., 35, 377–391 (2008). C.P. Kurtzman,Management and Genetic Characterization of Agricultural and Biotechnological Microbial Resources, Accession Numbers 408614 and 413230. Microbial Genomics and Bioprocessing Research Unit, USDA, ARS, NCAUR (2009). L.R. Lynd, W.H. Van Zyl, J.E. McBride, and M. Laser. Consolidated bioprocessing of cellulosic biomass: an update. Curr. Opin. Biotechnol., 16, 577–583 (2005). National Institutes of Health. NIH guidelines for research involving recombinant DNA molecules. April 2002, DHHS/NIH Bethesda, MD (2002); NIH_Guidelines_Apr_02.htm R.D. Perlack, L.L. Wright, A.F. Turhollow, R.L. Graham, B.J. Stokes, and D.C. Erbach.Biomass as feedstock for a bioenergy and bioproducts industry: the technical feasibility of a billion-ton annual supply. A joint study sponsored by U.S. Department of Energy and U.S. Department of Agriculture (2005). Renewable Fuels Association. Ethanol industry statistics; statistics. Accessed 25 January, 2009. United States Department of Agriculture, Animal and Plant Health Inspection Service. Biotechnology, Permits, Notifications, and Petitions (accessed 27 February, 2009); biotechnology/submissions.shtml. United States Department of Agriculture Animal and Plant Health Inspection Service. USDA-APHIS Biotechnology Regulatory Services User’s Guide: Notification (5 February 2008); http://www. United States Department of Agriculture Economics, Statistics and Market Information System. World agricultural supply and demand estimates (WASDE), March 11, 2009; http://usda.mannlib. A.J.A. Van Maris, D.A. Abbott, E. Bellissimi, J. van den Brink, M. Kuyper, M.A.H. Luttik, H.W. Wisselink, W.A. Scheffers, J.P. van Dijken, and J.T. Pronk. Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: current status. Antonie van Leeuwenhoek., 90, 391–418 (2006).

Advanced Biorefineries for the Production of Fuel Ethanol
Stephen R. Hughes, William Gibbons and Scott Kohl



Although the basic process of ethanol production has not changed greatly for several decades, significant improvements were made throughout the 1980s and 1990s in the design and engineering of fuel ethanol production facilities. Prompted by the oil crises of the 1970s, Brazil expanded its production of sugar cane ethanol with the Proalcool program (Nass et al., 2007), whereas the United States launched a fuel ethanol program based on corn (Bothast and Schlicher, 2005). An additional impetus for US ethanol production occurred during the late 1990s, when the commonly used oxygenate methyl tert-butyl ether (MTBE) was banned due to groundwater contamination. Subsequently, the demand for ethanol rose dramatically in the early 2000s and fuel ethanol plant construction boomed. Growing concerns worldwide about dependence on imported oil, the depletion of fossil fuels, and environmental pollution brought about new policies in biofuel production. The European Union (EU) is moving from the target of 5.75 % by 2010, as proposed under directive 2003/30/EC, towards a mandatory biofuel usage of 10 % of the energy used for transport by 2020 (FAPRI , 2008 Agricultural Outlook). Japan has set a goal of replacing 20 % of its oil demand with biofuels or gas-to-liquid fuels by 2030 (Eikeland, 2007). Likewise, in 2005, the US enacted the Renewable Fuel Standard (Energy Policy Act of 2005) that required the use of 7.5 billion gallons (BG) of biofuels for transportation by 2012. Furthermore, a comprehensive legislation signed into law in December 2007 in the US mandated a phase-in of all renewable fuels, including conventional (corn starch)
Biomass to Biofuels: Strategies for Global Industries Edited by Alain Verts, Nasib Qureshi, Hans Blaschek e and Hideaki Yukawa The contribution of Hughes has been written in the course of his official duties as US government employee and is classified as a US Government Work, which is in the public domain in the United States of America.


Structure of the Bioenergy Business

Figure 4.1 Biofuels production is scheduled to increase significantly in the next decade, driven by total renewable fuel requirements as mandated by the US Renewable Fuel Standard. After 2015, ethanol production from corn starch should remain steady, while production from sugar cane will rise slightly, providing a slight increase in worldwide conventional ethanol production. The majority of supply growth of renewable fuels is expected to be met with advanced biomass-derived fuels, including biodiesel and cellulosic ethanol (FAPRI, 2008; ACE, 2008)

ethanol, advanced biofuels (ethanol and biodiesel from biomass), cellulosic biofuel, and renewable diesel, from a total of 9 BG in 2008 to 36 BG by 2022 (Energy Independence and Security Act of 2007; Figure 4.1). The corn-based ethanol industry in the US expanded from 110 plants, with a production capacity of 5.5 BG per year in January 2007, to 134 plants and 7.2 BG per year in January 2008 (Renewable Fuels Association). With higher commodity prices and ethanol distribution issues having impacted upon the pace of expansion (Krauss, 2007), the higher oil prices are likely to drive towards greater ethanol use and an increased production capacity worldwide (FAPRI, 2007; FAPRI, 2008; see Figure 4.1). At present, the cost per gallon of ethanol produced from sugar crops is competitive with the cost per gallon of gasoline ($2–3) and, indeed, in Brazil sugar cane ethanol has overtaken gasoline as a transportation fuel (FAPRI, 2008). Although the technical challenges remain, the need to identify a sustainable transportation fuel source will dictate the development of advanced biorefineries producing ethanol from cellulosic feedstocks at a competitive price. Design improvements have greatly increased the efficiency of new US biorefineries compared to older plants (Wheals et al., 1999). Between the 1980s and 2008, yields have increased from 2.5–2.6 gallons ethanol per bushel of corn to 2.7–2.8 gallons per bushel (Bothast and Schlicher, 2005), whereas energy inputs to produce one gallon of ethanol and dry coproducts have fallen from 67 768 Btu to 45 802 Btu (Shapouri et al., 1995; Shapouri et al., 2004); likewise, water use also has decreased from 5.8 gallons water per gallon ethanol to 4.2 gallons (Phillips et al., 2007). A plant built today with the latest technology can operate with 2.8 gallons of ethanol per bushel of corn, 20 000 Btu for ethanol

Advanced Biorefineries for the Production of Fuel Ethanol


production, 12 000 Btu for coproduct drying, and 3.5 gallons water per gallon ethanol (Zhang, 2006). Additional improvements to production have come from the development of high-yield corn, optimized enzymes and yeast strains, and higher-value uses for nonfermentable coproducts (Bothast and Schlicher, 2005). Further technological improvements are needed to address the remaining challenges, however. For dry grind plants these include long lag times at the start of batch fermentation (Vriesekoop and Pamment, 2005), the recovery of oil from byproducts, and zero water discharge requirements (Parkin et al., 2007). Dry mill plants are designed to fractionate the corn kernel into germ, fiber, and endosperm fractions prior to fermentation. Although this reduces the load of nonfermentable materials in the conversion process and recovers foodgrade corn oil, the achievement of high fractionation efficiencies can be challenging (Bothast and Schlicher, 2005). Wet mill plants usually employ steeping, which sacrifices some ethanol yield for the advantages of recovering oil, fiber, and protein fractions. Purified starch from wet milling is used for food applications, as well as the conversion to ethanol. Microbial contamination, and the resultant need for antibiotics (Skinner-Nemec et al., 2007), is a concern at both dry grind and wet mill plants. Commercial fuel ethanol production facilities have characteristic populations of bacterial contaminants that reduce the product yield and are difficult to eradicate (Skinner-Nemec et al., 2007). The US FDA has approved one antibiotic for use in the ethanol fermentation phase at a maximum of 2–6 ppm; however, the cost for that antibiotic at an ethanol plant producing 50 million gallons per year is approximately US$ 200 per dose in each fermentor (Phibro). In the long term, however, it is unlikely that the supply of corn and sugar cane feedstocks can fully meet all future desired renewable fuel needs in an economically and environmentally sustainable manner. Cellulosic biomass has great potential as a plentiful feedstock to allow expanded biofuel production for the future. A joint US Department of Energy/US Department of Agriculture (USDOE/USDA) study has estimated that over one billion tons of biomass can be sustainably produced in the US for biofuel production (Perlack et al., 2005). Brazil’s extensive history with ethanol biorefineries has attracted interest from scientists, producers and governments (Nass et al., 2007) and, in theory, expanding the Brazilian ethanol program by devoting all available cultivated land to sugar cane would produce enough ethanol to replace 10 % of the gasoline used worldwide (Goldemberg, 2007). However, in order to be fully efficient, an integrated system – similar to that of the petroleum industry – must be developed for the production of fuels from biomass (Cardona and Sanchez, 2007). This system must include the growth and utilization of low-cost biomass from all sources, the design of processing methods to obtain high yields of liquid fuels, and the production of other higher-value products. These biorefineries are expected to use engineered microbes, to integrate fermentation processes to utilize waste streams, to improve production efficiency, to recycle water and energy, and to reduce emissions of various pollutants.


Ethanol Production Plants Using Sugar Feedstocks

Sugar cane, sugar beets, and sweet sorghum are sugar crops used as feedstocks for ethanol production. Their chief advantages are a high yield of sugar per acre, and low conversion


Structure of the Bioenergy Business

costs; for example, the total cost of ethanol production from sugar cane in Brazil is estimated as US$ 0.81 per gallon (Shapouri et al., 2006). The main disadvantage of using these crops is their natural seasonal availability. In Brazil, 10 % of the total cultivated land (i.e., 13.8 million acres) is devoted to sugar cane, with about 7.4 million of these acres being used to produce 4.2 BG of ethanol (Goldemberg, 2007). Sugar cane is a particularly costeffective source of biofuel, since the fiber in the stalks and leaves (bagasse) is used to generate process steam and electricity for the biorefinery. In addition, the liquid effluent (vinasse) is used as a fertilizer and irrigation supply to the cane fields, thereby eliminating costs for wastewater treatment (Kojima and Johnson, 2005). Brazil’s ethanol production is the cheapest in the world, costing about US$ 0.81 per gallon (without import tax) (2006 data; Goldemberg, 2007; Shapouri et al., 2006). Sugar cane must be processed within 24–72 h of harvest. The sugar is first isolated by crushing the stalks with specialized rollers to release the juice (Figure 4.2). Lime (calcium hydroxide) is then added to precipitate the fiber and sludge, and the mixture is then filtered.

Figure 4.2 Schematic representation of sugar cane biorefinery, showing the value-added sugar and molasses feed coproducts. ‘A’ molasses is the liquid remaining after 77% of the raw sugar in cane juice has been extracted during the first crystallization stage. Further processing of the ‘A’ molasses extracts another 12% of the raw sugar to yield ‘B’ molasses, that is used for subsequent fermentation to ethanol. Invertase, which hydrolyzes sucrose, is added to augment yeast invertase. Dextranase prevents the bacterial formation of dextran polymers that impede sugar recovery. In Brazil, the hydrous ethanol (5% water) resulting from distillation is used directly for fuel

Advanced Biorefineries for the Production of Fuel Ethanol


The filtrate solution is evaporated to concentrate and crystallize the sugar, prior to its removal by centrifugation. The noncrystallized sugar and accompanying salts are concentrated to form a syrup called ‘blackstrap’ (B) molasses, that is used as a raw material for subsequent fermentation to ethanol. In the fermentation of molasses it is important to use a thermotolerant strain of Saccharomyces cerevisiae, since considerable heat is generated in the process. The maximal growth of S. cerevisiae is attained at 34–37  C, whilst at temperatures above 44  C the ethanol production is reduced and the residual sugar content is increased (Laluce et al., 2002). Notably, molasses is often contaminated by Leuconostoc mesenteroides, a bacterium that forms dextran chains from sucrose, thus necessitating treatment with dextranase (Jimnez, 2005). e Today, significant research is under way to expand sugar cane biorefineries through the processing of bagasse for cellulosic ethanol (Gnansounou et al., 2005). This strategy has a significant potential for improving the economics of sugar cane plants, as it may allow refineries to produce ethanol during the winter months when the sugar cane harvest season is over. The Brazilian biorefinery program serves as a model in several ways. First, the starting material is extremely cost-effective (Shapouri et al., 2006). Second, the output can be quickly and cheaply adapted to changing market demands. Third, the process is continually optimized through research and development, as exemplified by the significant economies of learning already achieved by this industry (Goldemberg et al., 2004). Sugar beets represent a major source of sugar in Europe and North America, and are also used for biofuel production in France. Sugar beets generate good yields (25–50 tons per acre) and grow in temperate climates with a lower rainfall than is needed for sugar cane. On average, the ethanol yield is 25 gallons per ton of sugar beets (Shapouri et al., 2006); however, ethanol production from sugar beet requires a greater chemical and energy input and consequently is more expensive than the process using sugar cane. Sweet sorghum varieties are few in number and not widely grown, although some have a significant sucrose content (500 gallons syrup per hectare). In sweet sorghum the sugar is contained in the main stalk, and is recovered by pressing the stalks with rollers (similar to the process used for sugar cane). Yields, on average, are 20 gallons of ethanol per ton of stalks (Gnansounou et al., 2005). China is currently seeking to switch from corn to sweet sorghum as a feedstock for ethanol, since sorghum is more drought-tolerant and can grow in the arid regions of China where corn does not perform well. An additional benefit is that the farmers can use the sorghum grain, since the ethanol is produced from the sweet juice in the stalk. At present, existing biorefinery facilities in China are being converted from corn-based to sweet sorghum-based ethanol production (Consultative Group on International Agricultural Research, 2007). These facilities combine new and established technologies for obtaining ethanol, sugar, grain, and fiber. The most profitable option is a flexible facility capable of serving both sugar and fuel ethanol markets, depending on their relative market prices. However, balancing the use of bagasse between ethanol production and power generation presents important engineering optimization opportunities (Gnansounou et al., 2005).


Dedicated Dry-Grind and Dry-Mill Starch Ethanol Production Plants

Corn is the second-largest feedstock source for ethanol production worldwide, and by far the most important starch feedstock. In 2007 worldwide, 11.6 BG of ethanol were produced

saccharification occurs whereby the dextrins are converted to fermentable sugars. After saccharification. Corn has a longer shelf-life than sugar crops. water-soluble glucose polymers) by adding water. density separation and washing steps to separate the fiber. A dry-milling process. Biofuels International. and nutritional composition of the coproducts (Applegate et al.. 2008). primarily corn gluten feed and corn gluten meal.’ Starch refineries use one of three methods for the initial processing of corn grain: . and starch. After separation.. and holding the mixture at temperatures of 90–100  C for 15 to 60 min. The use of corn starch as a feedstock. A dry-grind process. with significant starch processing facilities of all types throughout the world. however. the aim being to increase the quantity. Germany. sorghum has a short growing season. the cereal used to produce ethanol is wheat. 2009).8 (Olempska-Beer et al. The ground corn is then slurried in water with alpha-amylase enzyme and cooked at 85 to 140  C. followed by screening stages to separate the germ. . which are shorterchain.85 gallons per bushel of corn is achieved with a particle size of about 0. Although dry-grind/dry-mill plants (Figure 4. first to break down the cell wall that contains the starch. Corn-based ethanol production is concentrated in the US Midwest. respectively (Knight. which involves pretreating the kernels with sulfurous acid to swell them prior to a sequence of milling. the starch is liquefied (i.76 Structure of the Bioenergy Business from 2 billion tons of corn (Renewable Fuels Association. For the process of starch conversion. adds another step to the ethanol production process. 2005). In the dry-grind process. .3) cost less to construct and operate than wet-mill ethanol plants. which subjects the kernels to series of roller mills. fiber.. 2008. the corn is passed through hammer mills that grind it into fine particles so as to facilitate the entry of water and enzymes in the next steps of the process. in Spain. the United Kingdom and Canada. . Processing modifications to traditional dry grind plants to recover any nonfermentable fractions such as the germ. An optimum ethanol yield of about 2. France. A wet-milling process. recombinant optimized alphaamylases from Bacillus subtilis and Bacillus licheniformis are used that are stable at temperatures up to 105  C and operate best at pH 5. In addition. alpha-amylase enzyme. lipid.. yeast and nutrients are added and the mixture is first fermented and then distilled. The ethanol that emerges from the distillation column is passed through a molecular sieve to remove any remaining water. rye and barley are used in Germany and Spain. 2006). passing through a 2 mm screen (Rausch et al. the latter process yields more valueadded coproducts. germ. because the starch polymer must first be broken down into the constituent simple sugars in a process called ‘saccharification. In addition. hydrolyzed to dextrins. protein. and endosperm. which involves grinding the kernels into a relatively fine powder... On the other hand. A current concern is that the emergence of antibiotic-resistant bacteria may limit the effectiveness of the antibiotics used in ethanol-production facilities (Bischoff et al.e. such that the ethanol production plants will be idle for part of the year. 2006). quality.6 mm. and then to convert the starch polymer to dextrin oligomers. for example sorghum must be processed immediately after harvest as it loses over 20 % of its fermentable carbohydrates on storage. in this way. 2007). A temperature higher than 100  C will enhance the hydration and gelling of the starch granules and help to reduce contamination by (mainly) lactic acid bacteria (Skinner-Nemec et al. and fibrous portions of the corn kernel have been (and continue to be) developed. 2007).

the yeast is added as an inoculum and subsequently reproduces rapidly to produce ethanol and carbon dioxide in equimolar amounts. As the fermentor is being filled. due to its high ethanol productivity and tolerance and its robustness under industrial processing conditions (HahnH€gerdal et al. the anhydrous ethanol is denatured with a small amount of gasoline to produce fuel-grade ethanol. During the fermentation process (which is exothermic). Fermentation of the mash to produce ethanol is accomplished using S. it is necessary to cool the fermentor so as to maintain the temperature at 28–32  C and achieve optimum . This step uses the enzyme glucoamylase. and to minimize the risk of contamination. In order to minimize corrosion at the pH range used. Yeast is the most desirable organism for ethanol production from corn. a further enzymatic hydrolysis of the dextrins (saccharification) is required to produce the fermentable sugars. 2008). which liberates glucose molecules from the nonreducing end of the dextrin molecule.Advanced Biorefineries for the Production of Fuel Ethanol 77 Figure 4.3 Schematic representation of a dedicated dry-grind ethanol production plant. Glucoamylases exhibit maximum activity at temperatures of 60–65  C and at a pH of about 4. 2006). Saccharification adds to the cost of processing starch compared to sugar-based fuel ethanol production. the fermentation vessels are constructed from well-passivated carbon steel or 304 stainless steel. in most dry-grind plants the glucoamylase is injected directly into the fermentor using a process called ‘simultaneous saccharification and fermentation’ (SSF) (Warner and Mosier. The fermentation is typically conducted in a batch process that lasts for 48–72 h. After dehydration using a molecular sieve to remove all water.5. Large-scale industrial fermentation processes typically result in a ethanol concentrations of 12–15 % on a volume basis (Zhang. In order to reduce the capital costs required for saccharification vessels. cerevisiae. 2007).. Although the dry-mill process is usually a batch process. it can be run continuously if several tanks are employed and scheduled so that log-phase growth occurs in one tank as another is producing ethanol in the beer well After liquefaction.

Finally. the fermented broth is fed to a distillation system to recover the ethanol. A portion of the liquid from the centrifugation (thin stillage) is recycled to replace 10–40 % of the water needed to conduct the next fermentation batch. The syrup and wet cake are combined and dried to about 10 % moisture to produce dried distillers grain with solubles (DDGS). 2009). Although allowing a greater fermentor productivity. 2001).. either by passing water through a cooling coil contained within the fermentor. providing anhydrous ethanol prior to denaturation with gasoline for the production of fuel-grade ethanol. an animal feed supplement containing approximately 28–32 % protein. The distillation residue. The ethanol/steam vapor is then passed through a rectification column to produce an azeotropic mixture of 95 % ethanol: 5 % water. usually penicillin and virginiamycin (Skinner-Nemec et al. Water recycling and minimizing plant emissions are critical in plant operation. where the heat is transferred to water before the mash is returned to the fermentor. has voiced concerns about this practice (Juranek and Duquette. The solids produced after centrifugation are known as ‘wet cake’ or wet distillers grains (WDG). The heat removed can then be used elsewhere in the plant. between eight and ten fermentors are used. The trays in the stripping columns are designed to avoid becoming plugged with solids. With a correct sizing and scheduling of the fermentors. However. dry milling yields 2. 2006). the heat must be removed continuously. but to allow the steam to strip the ethanol from the beer. A molecular sieve dehydration (USDOE Genomics: GTL 2007) process is used to remove the last of the water. Consequently. approximately 12 000 kJ kgÀ1 ethanol is generated (Kwiatkowski et al. with a total retention time in each set of 48–72 h. Most modern ethanol plants have zero wastewater discharge. Typically. and efforts are being made to achieve zero net water usage by developing anaerobic digesters or . which has regulatory authority over stock feed ingredients. or by pumping the fermenting mash through a large heat-exchanger. 2007). staggered in overlapping sets of three. In order for the process to continue optimally. 2005). which is known as ‘whole stillage’ and contains all of the unfermented nonvolatile components. Thin stillage also recycles any nutrients from the yeast cells that have been lysed during distillation and cooking back to the fermentation step. After fermentation.. and yeast strains that express antibacterial peptides (Bischoff et al. including chemical agents that kill bacteria but not yeast. Typically. and exhibit a typical moisture content of 60–70 %. the mash is rapidly transferred to a beer well (Figure 4. continuous fermentation processes require much greater care to prevent contamination from spreading throughout the system (Bayrock and Ingledew. new methods are being developed for microbiological control. is sent to continuous centrifuges. but the remainder of the thin stillage is sent to an evaporator to produce a syrup that contains 50–60 % moisture. The quality and quantity of wastewater discharge or gas emissions are governed by both State and Federal regulations.3). thermotolerant yeast strains to allow operation at higher temperatures to impede the growth of contaminating bacteria. the upstream starch conversion steps and downstream distillation steps can be operated in continuous fashion. Center for Veterinary Medicine. 2007).8 gallons of ethanol and about 8 kg of DDGS per bushel of corn (Bothast and Schlicher. the US Food and Drug Administration.. which allows the fermentor to be quickly cleaned and re-used in the next batch. Microbial contamination during fermentation has been controlled for decades by the addition of antibiotics.78 Structure of the Bioenergy Business yeast performance. In terms of heat production.

4). but produces a range of higher-value products including gluten feed. although some is used as an animal feed supplement or fermentation nutrient. the corn oil can be used with new continuous column technology to produce valuable coproducts. the nonstarch fractions are still used as livestock feed. During steeping. and 7 kg of DDGS (Shapouri et al. germ. In some cases. but in others they are used for human foods or foodgrade zein polymers. Attrition mills remove the germ from the kernel during the first milling stage. the free sugar present in the corn is converted by bacteria to lactic acid. as well as fuel ethanol (Figure 4. 2008). for the production of dry ice. dry-mill facilities fractionate the corn kernel at the start of the process by using coarse mills and screening.4 Dedicated Wet-Mill Starch Ethanol Production Plants The wet-milling process is more complex and capital-intensive than the dry-grind process. New DDGS fractionation technologies will allow biorefinery products diversification by allowing the removal of oil for biodiesel production and/or fiber recovery for cellulosic ethanol. As an alternative to fractionating DDGS. the lactic acid and sulfur dioxide cause the kernel to soften such that the disulfide bonds are cleaved. the corn is conveyed in a continuous process to large tanks (steeps).Advanced Biorefineries for the Production of Fuel Ethanol 79 other water-treatment methods to recycle water (Pate et al. to serve as chemical feedstocks for the polyester and thermoplastic industries (Varadarajan and Miller. is concentrated by evaporation to 40–45 % solids. On the other hand. gluten meal. the mixture is then held at 49–53  C for between 22 and 50 h. After steeping. The carbon dioxide produced during fermentation is sold as an industrial gas. Higher-value coproducts will improve the economics and energy balance of the traditional dry-grind process. fiber. Most of the CSL is mixed into gluten feed. In wet-milling. corn steep liquor (CSL). the corn is separated from the steep water and processed through a series of several milling operations. Major ethanol companies are considering processes to remove oil from DDGS by using hexane to produce a higher-protein DDGS animal feed. The emissions of volatile organic compounds. breaking the key linkages between the starch. The profitability of starch-based biorefineries will ultimately depend on the value of all products manufactured. For example. primarily from the driers. have been greatly reduced by the use of thermal oxidizers (Phillips et al. corn oil and germ. where steep water containing 1–2 % sulfur dioxide is added. 2006). and thus permit the production of ‘designer’ feeds for specific animals. 8 kg of carbon dioxide. This softening process is aided by swelling of the kernel as a result of the water uptake.. becoming corn steep liquor (CSL). highfructose corn syrup. The steep water.. 4. 2007). Low-oil DDGS can be used at higher rates for cattle feed. such as diols and diacids. while low-fiber DDGS is more suitable for poultry or swine rations. a 25 kg bushel of corn yields approximately 8 kg of ethanol. 2007). which contains the corn solubles. such that the moisture content of the corn increases from 15 % to 45 %. and zein. The liberated germ is separated from the other components by density differential using . whereas gluten meal generally has a 60 % protein content and is used to feed poultry and swine. Gluten feed is generally a low-value feed and is sold on the basis of its protein content (18–22 %).. and to grow algae for biomass for ethanol and biodiesel production.

. The germ is purified through countercurrent washing before being de-watered and dried to less than 5 % moisture. so as to take advantage of economies of scale. so the fermentation can run in a continuous cascade. that are then removed through another screen step. Recycling eliminates the need for yeast to go through a log phase. Generally. Often. the pH of the starch slurry is adjusted to 5. the wet-mill process is very similar to that of dry milling. The starch. When the purified starch slurry has been obtained. Calcium is often added (20–100 ppm) to enhance enzyme stability. solid slurries of 30–40 % starch are common. The remaining components pass over a coarse screen to remove the fiber particles from the liquid flow.2 with lime. After dehydration using a molecular sieve.80 Structure of the Bioenergy Business Figure 4. and are sent directly to a high-speed centrifugation step. and the starch fraction is finally purified with countercurrent washing water through a series of hydrocyclones. First. As the starch stream is relatively free of fiber or other components. The coarse fiber fraction is ground to liberate any remaining starch and gluten. it is well suited to the high temperature and short time of jet cooking and subsequent enzyme liquefaction. after which alpha-amylase is added to convert the starch polymer into soluble short-chain dextrins (liquefaction). the anhydrous ethanol is denatured with a small amount of gasoline to produce fuel-grade ethanol hydrocyclones.8–6. Food-grade yeast can be sold as a feed additive or recycled into fermentatin.4 Schematic representation of dedicated wet-mill ethanol production plant using continuous fermentation showing high-value coproducts. before the fiber is dried. gluten and fine fiber fraction pass through the screens with the water. Hence. several facilities will pool their germ batches for processing at a central oil extraction plant. it is mixed with other components such as CSL to create coproducts such as gluten feed. A high-speed centrifugation is used to separate the gluten from the starch.

5 Schematic diagram of an advanced biorefinery capable of producing high-value coproducts (specialized animal feed. The final product from a continuous process will have an ethanol content of 8–10 % by volume.5). 4. EXOGLUCANASE. depending mainly on the degree of saccharification prior to fermentation. GASIFICATION. Assuming the development of cost-effective production facilities (Figure 4. DEXTRINASE. so as to achieve a sustainable coproduction of biofuel and electricity .Advanced Biorefineries for the Production of Fuel Ethanol 81 The slurry from the liquefaction stage is mixed with heat-sterilized steep water and sent for saccharification. SWITCHGRASS ENZYMES. (A) SWITCHGRASS. After saccharification. PEROXIDASE. and on engineering a multifunctional recombinant ethanologen to ferment the sugars in the biomass.4). OIL PLANTS WITH LIGNOCELLULOSE Lignocellulose Biomass Densifying Chopping Fine Grinding (B) PLANT OIL REMOVAL FROM LIGNOCELLULOSE ETHYL ESTER BIODIESEL CELLOBIASE. Most wet mills practice continuous-cascade fermentation (Figure 4. ESTERASE Heated Biomass Hydrolysate Production Neutralization POLYESTER THERMOPLASTICS TRANSESTERIFICATION UTILIZING CATALYTIC COLUMN Wet Mill Corn Steeping Grinding Filtration Washing Germ Separation Grinding Separation Liquefaction Starch Cellulase Hemicellulase Enzymatic Treatment Lignin Separation Liquid Hydrolysate Enzymatic Saccharification SSF Yeast Fermentation Distillation Dry Mill / Grind 1. PECTINASE. ARABINOFURANOSIDASE. ALPHA AMYLASE. LACCASE. The steep water provides both the fermentation nutrients and pH adjustment for saccharification. BETA AMYLASE.3-PROPANEDIOL Enzymatic Saccharification Yeast Fermentation Beer Well CORN OIL Liquifaction Enzymatic Saccharification Yeast Fermentation Distillation ETHANOL THERMOCHEMICAL CONVERSION. S. ENDOGLUCANASE.5 and a temperature of 65  C. which facilitates yeast recycling and improves the overall fermentation rates. The primary advantage of a combined biorefinery is a reduction of costs by sharing energy and manpower. biodiesel.5 Dedicated Cellulosic Ethanol Production Plants Supporters of renewable biofuels view ethanol produced from lignocellulosic feedstocks as the type of biofuel that has the greatest potential for providing renewable energy for the future. BIOCRUDE THIN STILLAGE Distillation Dehydration ETHANOL Dehydration ADVANCED NUTRITION THROUGH TAILORED ANIMAL FEEDS USING RECOMBINANT YEAST Wet Distillers Grains (WDG) Dried Distillers Grains Solubles (DDGS) GERM FIBER GLUTEN Figure 4. BETA XYLOSIDASE. The profitability of the lignocellulosic process depends on obtaining enzymes for deconstructing the biomass. bioethanol). PROTEASE. BETA GLUCOSIDASE. cerevisiae is added to ferment the sugars to ethanol and CO2. in which the added glucoamylase converts the dextrins to glucose at a pH of 4. PYROLYSIS.3-PROPANEDIOL CONDENSED WITH LIGNOL AROMATIC DIACID Corn Milling Cooking SSF RECOMBINANT SACCHAROMYCES CEREVISIAE ANAEROBIC PRODUCTION 1. combining lignocellulose-based (A) with corn-based (B) ethanol production. biothermoplastic. Very few insoluble solids are found in these fermentation systems. XYLANASE. The total fermentation time will be between 20 and 60 h.

Overall. the new plant is designed to provide power to an adjacent corn starch dry-grind mill (USDOE Biomass Program Deployment. a panel of experts from the Army Natick laboratory anticipated that the release and fermentation of glucose from cellulose would be possible by 1980. milo stubble. with pelleting and briquetting having been applied to switchgrass. In contrast.5. wood residues. The aim of this ambitious project is that the proposed plants will each use from 700 to 1200 million tons per day of cellulosic feedstocks. and hydrolyze the cellulose and hemicellulose components into their constituent monosaccharides (Saha. petroleum prices sharply decreased when the oil shock ended. Another requirement is the availability of organisms that exhibit the major process robustness required for the simultaneous . 2007). 2005). which in turn resulted in a sharp decrease in the budgets allocated to research aimed at developing biomass ethanol. 2003). A recent initiative that is particularly worth noting is that of the US Department of Energy that awarded funding to six companies to develop commercial-scale biorefineries. wheat straw. the technologies involved in this process are more complex than those in existing ethanol production plants (Vermerris et al. A wide range of countries have adopted programs for increasing the contribution of ethanol from biomass to their transportation fuel supplies (Kojima and Johnson. rice straw. This is particularly exemplified by a USDOE/USDA study (Perlack et al. Several of these plants are designed to generate sufficient energy to cover their energy needs.4 to 125 million gallons of ethanol annually for each of the plants. of stimulating rural development. During the 1970s. remove the lignin. during which it was calculated that the US has an annual production capacity of 368 million tons of forest biomass and 998 million tons of agricultural lignocellulose.. 2005). making the hemicellulose and cellulose accessible to enzymatic treatment. and decreasing energy supply risks. benefiting the overall economy in developing countries. and wood-based energy crops. Production estimates range from 11. 2007). energy grasses and municipal bioproducts can involve a variety of chemical methods (Varga et al. The conversion of lignocellulosic biomass into ethanol (Figure 4. corn fiber. wood and vegetative wastes. green and wood landfill waste. In one case. 2003). including corn stover. Field densification techniques for agricultural materials are also important for minimizing transportation costs. alkaline methods typically depolymerize the lignin. It is striking that only recently has funding become more broadly available again for conducting research in this area (USDOE Biomass Program Deployment. since biomass has a low bulk density. Biomass pretreatment of agricultural residues. with the explicit purpose to use cellulosic feedstocks. part A) requires pretreatment to break down the lignocellulose structure. and shelled corn (Doering and Morey.. 2007). However.. 2007). while this aim was perhaps technically achievable. The use of biomass for ethanol production is attracting interest worldwide as a means of reducing greenhouse gas emissions. yard. switchgrass. for a total annual availability of 1366 million tons. corn stover. cobs and stalks.. The sugars released by hydrolysis of cellulose and hemicellulose are then converted to biofuels via fermentation (Van Maris et al. barley straw. Collection and transportation is a significant issue. Among the critical requirements for making the cellulosic biofuel vision a reality are the availability of efficient and costeffective cellulase and hemicellulase enzymes to hydrolyze pretreated cellulose and hemicellulose into fermentable monomeric sugars. 2006).82 Structure of the Bioenergy Business cellulosic biomass feedstocks could supply a growing ethanol industry with large quantities of less-expensive raw materials. Acidic methods generally depolymerize the hemicellulose and make the cellulose accessible to enzymatic treatment.

new farming practices as well as methods for its treatment are required. Upstream of this value chain are the farmers who need to be provided with the appropriate economic incentives to grow bioenergy crops. In the case of ethanol production (Figure 4. economies of scope. Unfortunately. the use of forest thinnings and wood crops. which typically offers synergies including economies of scale. 4. namely its more corrosive nature and natural affinity for water. smaller and fewer process units. for example. 2008). In particular. since lower concentrations of ethanol are still. and which can utilize both hexose and pentose mixtures. requires new technologies.Advanced Biorefineries for the Production of Fuel Ethanol 83 and efficient fermentation of the resultant hexoses and pentoses. . 2007). Moreover. as well as of pulp and paper waste streams. such as gasification or pyrolysis. additional high-value uses of these compounds are envisioned in the future. this translates particularly into reduced energy costs. combined biological and downstream steps. and the promise to reap faster learning curve benefits. the dehydration of this so-called ‘ethanol broth’ is a major issue as it remains an energy-intensive process. for large-scale ethanol distribution – as is envisioned for cellulosic ethanol – the implementation of a pipeline system would provide significant savings in ethanol transport costs. allowing a considerable lowering of the enzyme costs. Handling of waste streams from thermochemical conversion is a further technical barrier (International Energy Agency.. 2006).5). in order to exploit manure for cellulosic energy. 2003. 2008) in laboratory trials. which in turn will reduce the energy inputs to be sourced from power stations. a major difficulty here is that such a pipeline system would need to be suited to the specific properties of ethanol. where it is blended into gasoline at up to 10 % by volume. 2007). significant investigations are still required to engineer industrial microbial strains for commercial use that are capable of rapidly producing and tolerating high ethanol concentrations at industrial temperature conditions. Wilkins et al. ethanol is currently distributed via rail car and trucks to specific areas. Gasification is a complex function for which varying levels of processing equipment must be incorporated. In addition. to this date. For example. China is constructing a 50 000 ton per year corncob process biorefinery at Yucheng (Qu et al. obtained when glucose and pentoses are utilized in the same fermentation batch.6 Advanced Combined Biorefineries Among others. A key concept here is that of process integration (Cardona and Sanchez. In the case of lignin. the US and South America are developing novel biorefinery designs for enabling a sustainable. The ethanol recovery techniques must also be improved. Notably. Similarly. However. Nevertheless. it is worth noting that lignin and other byproducts of ethanol production from biomass can be used to produce the thermal energy required for the distillation and dehydration steps. new facilities and equipment are needed to enable the use of municipal waste as a source of cellulose. a variety of useful phenolic derivatives can be generated (Kleinert and Barth. an infrastructure to collect and handle these crops must be created. and economies of learning. Despite dramatic improvements having been made in the development of cellulose-degrading organisms (Dien et al.. cost-effective process configuration for the biotechnological production of ethanol.. In the US. However. One critical issue that must be considered is that the production of large-scale cellulosic ethanol will only be possible if all links of this novel economic value chain are fairly rewarded.

with potentially lower feedstock costs and higher conversion efficiency (US EPA. cellulosic biomass and corn grain would be directly competing as feedstocks in the production of ethanol: approximately 30. and was based on the hypothesis that the main ethanol manufacturing projects under development will be completed successfully.e.. especially in conditions when the price of petroleum feedstock reverts to its low historical mean.. along with energy decentralization.7 Perspective The first step toward achieving a cost-effective. 2007).1.. A study was recently conducted to evaluate the ability of the agricultural sector to meet the goal of supplying 10 % of the US fuel demand with biofuels by 2020. A comparison of the actual costs and profits of dedicated ethanol production facilities versus the potential costs and profits of a combined biorefinery is presented in Table 4. The fact that only minor modifications to existing ethanol production facilities will be necessary gives high flexibility and favorable economic impact.84 Structure of the Bioenergy Business However. Cellulose can also be burned to supply the thermal energy that is required to power the biorefinery..32 billion bushels of corn would be used to produce 11. Notably. gasification plants constitute one of the breakthrough technologies in the biomass arena. 2007). Co-firing with a mix of biomass and coal or lignite is another approach.24 BG of ethanol. conversion technologies. Many manufacturing plants operate combined heat and power (CHP) systems in which a heat exchanger absorbs and converts exhaust heat into electricity via a generator. and simultaneously increased bioenergy and bioproduct demand quantities. using both corn grain and cellulosic biomass (De La Torre Ugarte et al. by 2011. Moreover. The main conclusion of a study that was conducted to address the goal to make cellulosic ethanol competitive with corn ethanol by 2012 was that indirect steam gasification (i.or lignite-only systems. existing technologies should be combined with new ones that have reached the stage of proof-of-concept. Growing environmental concerns. producing 16. smaller investments are required to implement CHP systems as compared to biomass-only plants. wood chips converted to ethanol by passing syngas over a fixed-bed catalyst) is a technology of choice for biomass conversion to ethanol (Phillips et al. an important consideration for ensuring the continuous funding of cellulosic feedstock biorefinery development in the world. and can reduce emissions by between 2 % and 25 %. 2007). As described in other chapters of this monograph. Gasification of biomass is essentially achieved by an endothermic technology that converts solid fuel to combustible gas.5 million dry tons of switchgrass and 2. the model used in this analysis combined commercially available subprocesses with expected biomass availability in 2012 (Perlack et al. drive this concept. to make this concept a reality. feedstock supply.73 BG of ethanol (De La Torre Ugarte et al.. as compared to coal. The results of the analysis. fully sustainable fuel ethanol biorefinery infrastructure is to demonstrate that lignocellulosic production plants are commercially . by which time almost half the projected ethanol demand would be expected to be met through the use of cellulosic biomass feedstocks. This approach decreases the technology risk. 2007). which incorporated agricultural market dynamics. The analysis extended only to 2014. 4. 32. 2005). projected that.. The study promoted the view that ethanol produced from biomass could be cost-competitive as compared to corn ethanol (Phillips et al. 2007).5 million dry tons of corn stover.

S. Particularly.00 mt Experimental 0. 2009. Furthermore.56 5. bioplastic monomers. A combined or crossover biorefinery will be the source of valuable coproducts. Eastern Regional Research Center.00 mt Experimental Potentially 0 >70 >100 Based on 2008 adjusted prices for construction materials.54 <0. . USDA Economic Research Service.46 mt 0. such as high-nutrient animal feeds. This product diversification strategy is critical for the long-term economic success of the biorefinery industry.46 mt 0. Expanding ethanol manufacturing to include lignocellulosic feedstocks as its primary feedstock with optimized recombinant yeast strains is expected to provide large quantities of fuel ethanol. In the long term – and assuming that a creative tension remains between the price of petroleum and bioethanol manufacturing costs – the production of ethanol from biomass is likely to become commercially feasible.84 >60 188.5 40–60 42.80 Sugar cane 62. USDA National Agricultural Statistical Service. 2008. multifunctional recombinant ethanologenic organisms and of custom-designed production processes.1 Comparison of costs and profits for dedicated versus combined or crossover refineries Ethanol refinery cost comparison 2008 Average cost of plant (US$ million)a Lifespan of plant (years)b Price of feedstock (US$)c Production costs (US$ per gallon)d Cost of enzymes (US$ per gallon)e Total ethanol profit (US$ billion)f Total coproduct profit (US$ billion)g a b c 85 Wet mill 233.05 Dry mill/ Grind 115. Time projections made at time of construction and expected amount of ethanol produced in planning stages. h For concept plant from using shared utilities and operational staff. Supergroup chemical engineering and cost evaluations of biofuels processes. 2009.. it will facilitate market segmentation and penetration.00 Continuous 95.71 0.88 0. or for wet and dry mill starch operations in Midwest United States. and biodiesel. as the overall manufacturing capacity of the ethanol industry grows. based on Chicago board of trade ethanol average price in third quarter 2008.77 9. including the creation of industrially robust.06 16. In turn. g For coproducts of representative plants used in study. Anonymous. Sources: Ramirez et al. d Cost for plant operation 2008 feedstock prices for sucrose operations in Brazil. viable.30 0.01 46. and it would enable one to capture greater overall value by reducing consumer surplus and to mitigate against oversupply risk.00 Continuous <50. energy. f Value represents world production levels for the year or theoretical production for year 2008 in US$.051 Experimental Crossover refineryh >200. USDA.64 Lignocellulose >375. ARS. transportation sector 2007. this will enable the flexible worldwide usage at the local level of all available biomass to establish a self-sustaining biofuel industry. Biofuels in the U. e At 2008 Novozyme and Danisco commercial price levels. Conservative prices for 2008 average price levels for first quarter.and manpower-sharing strategies will be used in the combined biorefinery to reduce costs. organic chemicals.65 6.Advanced Biorefineries for the Production of Fuel Ethanol Table 4. Realizing that the biorefinery vision will require an optimization of the entire biomass to the biofuel value chain.06 2. to reduce dependence on petroleum.00 mt 0.5 30–60 188.

E. B. Feed ingredient co-products of ethanol fermentation from corn. International Crops Research Institute for the Semi-Arid Tropics. Bayrock and W. and M. Maier. References ACE (American Coalition for Ethanol). News Release. polysaccharides. English. polyester fibers. and T. 117–122 (2009). biodegradable films. Institute of Agriculture. organic acids. BioEnergy. 39–45 (2001). Bischoff. 19–25 (2005). M. Cotta. United States Department of Energy. lignin will provide energy for heat and power. Anonymous.M. Biofuels International. Walsh. Worthington. T. Rich. while the nutrients in corn will balance the low nutrient value of lignocellulose. S.S. 103 (1). Official Energy Statistics from the U. Appl. and amino acids. June 13. In the crossover biorefinery.D. Hellwinckel. Energy Information Administration. Leathers. T. R. Feedstocks of the future. Available at: http://www.M. Acknowledgments Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply reccommendation or endorsement by the United States Department of Agriculture. Jeffries. Menard. Government. D. Changes in steady state on introduction of a Lactobacillus contaminant to a continuous culture ethanol fermentation. Bioresour. 27 (1). Schlicher. D. Biotechnol. Purdue Extension. Microbiol.. in addition to sweeteners. 258–266 (2003).W. Low-cost biofuel production in a crossover biorefinery will help to ensure a self-sustaining biofuel industry for the future.J. R. Ethanol from sweet sorghum does not compromise food security. Ingledew.ethanol. Knoxville. Microbiol. and bioproduct demands.A. C. B. Applegate.. Sanchez.J. De La Torre Ugarte. December 2006.J. Liu. June 2009. K. Fiber fractions from corn or sugar crops can be integrated into the lignocellulosic process and sugar concentrations increased by mixing sugars produced from all feedstocks. transportation sector. Biofuels in the U. TN: University of Tennessee. 2007.A. pharmaceuticals. February 2007.M.G. Bothast and M. or on land not used for food crops.O.. Journal of Industrial Microbiology and Biotechnology. including biodiesel. cost-effective biofuel production will require the generation of high-value coproducts in the biorefinery. biofuels. 2007. Richert. Dien. Cardona and O.C. R. Consultative Group on International Agricultural Research. C. Bioeng.. 63. Purdue University.A.E. B.S. D. and expanded uses for DDGS. . Department of Agricultural Economics. 2415–2457 (2007). corn oil. Fuel ethanol production: process design trends and integration opportunities. 25 September 2007. Economic implications to the agricultural sector of increasing the production of biomass feedstocks to meet biopower.86 Structure of the Bioenergy Business To achieve the goal of sustainable. Technol. Bacteria engineered for fuel ethanol production: current status. Public policy: federal legislation. diols. Farmers are now planting dedicated biofuel crops between food crop rotations. 67. Appl. Biotechnol. Biorefineries must be located close to the source of the biomass feedstocks to keep transportation costs to a minimum. and J. 98. Modeling bacterial contamination of fuel ethanol fermentation.S. Biotechnological processes for conversion of corn into ethanol.

Doering and V. B. Appl. Arraes Pereira.Ethanol’s boom stalling as glut depresses price. Barth. and M. I. Biofuels – challenges and opportunities.M. B. C.L. Pharmacol. Department of Economics. 2008. 110th US Congress. Parkin.. World Biofuels. Biofuels in Brazil: an overview. October 2005.L. e P. Refining sweet sorghum to ethanol and sugar: economic trade-offs in the context of North China. Kojima and T.S. M. 23. X.J. Weyer. C. A. New York Times. Norway.A. Workship. and C. Iowa State University. 96 (9). March 6. 985–1002 (2005). Einfield. Prod. 2007. C. E. Washington. 2228–2237 (2007). Iowa.B. Wright. . Department of Agriculture..S. Kwiatkowski.. Los Alamos. and T. 140–144 (2002). C. J. Dauriat. FAPRI Agricultural Outlook. Hightower. Department of Economics. 808–810 (2007). Distillers Grains Quarterly. Ethanol learning curve – the Brazilian experience. 2007. Regul. 30 October 2007.E. Toxicol. 20–27 (2005). Stokes. Duquette. Ditto. A. Biotechnol. and D. Just. Densification of biomass.J. K. Golden. Gorwa-Grauslund.Advanced Biorefineries for the Production of Fuel Ethanol 87 A. 2007. M. Spencer-Martins.. March 13. K. E. Modeling the process and costs of fuel ethanol production by the corn dry-grind process. Fonseca. Energy Sector Management Assistance Programme Report. 2007. Laluce. Managing High Energy Costs II: Discussion of Alternatives & Opportunities. Souza. Merker. L. Public Law 109-58. Energy Independence and Security Act of 2007.T. Goldemberg. Trondheim. National Renewable Energy Laboratory. Food-processing enzymes from recombinant microorganisms – a review. Bioresour. J. Center for Producer Owned Energy. and G. Innovation Management. F. P.J. McAloon.R. 47.C. 45. CO. 26. Coelho. World Biofuels. Energy Policy Act of 2005. Qu. Pate. Taylor. Zhu. Aden. Lin. O. Microbiol. E. Graham. Eggeman. 1. 2007. Dayton. Ind. Washington. G. Juranek and P. R. 2007. M.. Hahn-H€gerdal. 74 (5). and D. Ellis. S. Ames. Golden. UK (2008).I. Redwood Falls. P. Y. J. N: Sandia National Laboratories. M. Continuous ethanol production in a nonconventional five-stage system operating with yeast recycling at elevated temperatures. Walker. Goldemberg. Biotech. R... Krauss. Overview of energy-water interdependencies and the emerging energy demands on water resources. Trondheim Conferences. Kleinert and T. 29. Towards a industrial pentose-fermenting yeast strains. Johnson.M. J. L. Phenols from lignin. 6. Gattas. Thermochemical ethanol via indirect gasification and mixed alcohol synthesis of lignocellulosic biomass. P. Morey. A. Appl. and M. M. Colorado. Iowa State University. The International Bank for Reconstruction and Development/The World Bank. Food and Agricultural Policy Research Institute. DC.S. H. and C. Department of Energy and U. Potential for biofuels for transport in developing countries.F. B.L. Knight. Eng. Jechura. Phillips.. The dextranase along sugar-making industry. 736–745 (2008).A. Liu. Crop. Fourth Quarter. Iowa State Center. 301–304 (2004). J. Chem. Eikeland. April 25. Olempska-Beer. Biotechnol. 1235–1240 (2006). D. Report SAND 2007-1349C. Technical Report NREL/TP510-41168. 288–296 (2006). Technol. International Energy Agency Bioenergy: The Biorefinery Concept. Perlack. Riding the bioeconomy wave: smooth sailing or rough water for the environment and public health? Proceedings of the 2007 Iowa Water Conference – Water and Bioenergy. Technol. Lucon. R.R. Turhollow. 31. R. 30 September. 2007.R. Nass. Ethanol for a sustainable energy future. 937–953 (2007). Wyman. D. Minnesota.Biomass as feedstock for a bioenergy and bioproducts industry: the technical feasibility of a billion-ton annual supply. Microbiol.L. April 2007. Crop Sci. Cameron. Biofuels – their impact on crop production worldwide. Nastari.F. Jimnez. DiNovi. C. and W.. 109th US Congress. Science. J. 22. Antibiotic regulatory considerations for distillers grains. S. Ind. Studies on cellulosic ethanol production for sustainable supply of liquid fuel in China.S. 144–158 (2006). Erbach. and J. FAPRI Agricultural Outlook. Gnansounou.L. and O. Karhumaa. C. Food and Agricultural Policy Research Institute. P. Johnston. Biomass Bioenerg. Cambridgeshire. A joint study sponsored by U. Abud. 2005. Z. 315. Biotechnol.L. A. and D. DC.D. Bao.

C. S. Pronk.R. W.C. N.. Zhang. SunGrant. L. Shapouri. The economic feasibility of ethanol production from sugar in the United States. Biotechnol. Soc. The 2001 net energy balance of corn-ethanol..ers. Office of the Chief Economist. 47. Biochem. A. Miller. and T.S.Ethanol – dry grind process. DC. 4 August. Leathers. Microbiol. USDA/Economic Research Service. and H.D. R.R. Biomass Research and Development Initiative. IMB4. 37–50 (2003).E.H. TIBTECH. Acetaldehyde addition and pre-adaptation to the stressor together virtually eliminate the ethanol-induced lag phase in Saccharomyces cerevisiae. Office of Energy Policy and New Uses. 1995. Economic Research Service. R.S. E. D.C. Ind. Ramirez.W.A. Varadarajan and D.A. BioWeb. National Biobased Products and Bioenergy Coordination Office. Singh. Appl. B.E. 15 (5). A. Agriculture Economic Report No. Biofilm formation by bacterial contaminants of fuel ethanol production. June 2009.P.. 2007. and V. M. 23 April. Shapouri. Office of Biological and Environmental Research. J. van den Brink.N. Wisselink.. Ejeta. Antonie van Leeuwenhoek. 2006. 721.L.nass. and M. 29. Skinner-Nemec. Alves. 17. McAloon. Crop. and I. Prog. Wheals. and M.B. A.M. and M. United States Department of Agriculture/Baton Rouge. Fairbanks. Biotechnol. A.J. N. 12 November. H. Office of Energy. Abbott. E. Varga.N. Agricultural prices each year. USDA/Economic Research Service. J.E. J. 2007. Tumbleson. J.usda. Genomics: GTL (formerly Genomes to Life). 41. Scheffers. United States Environmental Protection Agency. van Dijken. 90. Available at: http://www.. Process Biochem.A. Pretreatment of corn stover using wet oxidation to enhance enzymatic digestibility. Mosier. United States Department of Energy and Department of Agriculture. and J. and N. Engineering process and cost model for a conventional corn wet milling facility. Available at: http://www.M. and IMB5 under anaerobic conditions.S.ethanolrfa. Washington. Wang. USDA/USDOE Report. W. 424–427 (2005). 43 (4). June 2009. 104. 279–291 (2003). United States Department of Agriculture. Trans. 28 Saballos. Fermentation of xylose by the thermotolerant yeast strains Kluveromyces marxianus IMB2. Reczey. Biotechnol.R. K. Vermerris. 30. Washington. Microbiol. Ind. Prod. Ellersieck. Thomsen. A. Amorim. Am. United States Department of Agriculture. Carpita. D. Basso. Biotechnol.usda.J. 2004.S. Mosier. 10 August. H. and A. Warner and N. L. Molecular breeding to enhance ethanol production from corn and sorghum stover. D. Salassi. Bellissimi. and J. Energy efficiency and renewable energy. Fuel ethanol production. M. 346–350 (2008). Rausch. http://www. biobased products and bioenergy initiative success stories. A.B. 48 (1). Louisiana State University. Lett.. Belyea. Eng. Hemicellulose bioconversion. F. M. H. Schmidt. A. Agric. Johnston. Particle size distributions of ground corn and DDGS from dry grind processing. Renewable Fuels Association.G.asp. Crop Sci. Nichols.. National Statistical Service. July 2006. 482–487 (1999). DC. 2008. Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: current status. Office of Energy. Appl.A. 27. 391–418 (2006). Vriesekoop and N. Singh. Van Maris. McAloon.D.88 Structure of the Bioenergy Business E. D. Combined Heat and Power Partnership. Shapouri. Eichling. 379–383 (2007). S. M. K. . 2008. Ladisch. M. Luttik.The US dry-mill ethanol industry. Pamment. 91–97 (2008). Duffield. Estimating the net energy balance of corn-ethanol. J.. United States Department of Energy Office of Science. 845–854 (2008). Fuel ethanol after 25 years. Banat. 2007. 273–277 (2005). Washington. M.C. United States Department of Energy. Biomass Program Deployment. J. Lett. K. H. S142–S153 (2007).T. Farm economy. Kuyper. A. W. G. Saha. V. Ethanol Fact Sheet. Johnston. Wilkins. Mueller.. Yee. Bioprocess engineering and biobased industrial products: catalytic upgrading of fermentation-derived organic acids.

Part II Diesel from Biomass .


Unfortunately. Nasib Qureshi. interest in fuel substitutes produced from feedstocks other than crude oil increased dramatically. Andrea Kruse and Klaus Raffelt 5. the combustion of. Biomass to Biofuels: Strategies for Global Industries and Hideaki Yukawa Ó 2010 John Wiley & Sons. requires high-quality standards to be met. The lower. inconsistent qualities of pyrolysis oils produced in the today’s processes. when the production of charcoal was less important economically.5 Biomass Liquefaction and Gasification Nicolaus Dahmen. compared to mineral oil-based products. interest in these processes is being revived. Hans Blaschek e . following the oil price crises during the 1970s. for example. This could imply simple and cheap fuel production facilities. it is important that only one key process step is required. In addition. Originally. Direct liquefaction is a challenging concept. This also hampers standardization of the products obtained in this way. Two principal routes can be followed: (i) direct liquefaction to fuel substitutes. To make such a process commercially competitive.1 Introduction The production of biofuels by thermochemical conversion routes offers a broad range of potential technologies. no commercial application was achieved in those days and R&D eventually was discontinued. Edmund Henrich. However. even on a small scale. the yield and composition of liquids produced may change drastically as a result of different feedstocks. by fast pyrolysis – was employed in the production of chemicals. and well-funded R&D activities in this field have been resumed. pyrolysis oils in engines and turbines. require substantial cleaning and grading-up for use in internal combustion engines. Today. However. or (ii) the production of high-quality synthetic fuels from synthesis gas generated by biomass gasification. Ltd Edited by Alain Verts. biomass liquefaction – for example.

lead to a logistics problem. . Germany Wilhelmshavener Raffineriegesellschaft mbH. Schwedt/Oder. www. three main routes can be considered industrially for the direct liquefaction of biomass to fuels for internal combustion engines: .miro-ka. Some pilot plants and first industrial applications exist for heat and power production from fast-pyrolysis-derived oils. Wilhelmshaven. However. Large-scale bio-synfuel production from poor lignocellulose materials (e. Hydrothermal liquefaction Pyrolysis Direct catalytic liquefaction. 5. biomass gasification and the subsequent production of synthetic fuels (diesel fuel.1.. As yet. Germany Exxon Mobile. bioliq. no examples are known of these fuels being used to power transportation vehicles. In this chapter. and examples provided of the most typical and most relevant processes in these two important synthesis steps. . dimethylether. The local distribution of biomass.pck. The production routes to synthetic fuels are described in detail in Chapter 6. Alternative liquefaction processes include . by using the Karlsruhe bio-slurry gasification 1 barrel per day %49. The annual fuel output capacities of modern oil refineries are in the 10 000 000 t range. On the other hand. some examples are listed in Table 5. economies of scale are essential to ensure the economic viability of biomass gasification plants. www. which must be solved in parallel with the technological process development necessary to allow feedstock with a high ash content to be used. Germany PCK Raffinerie GmbH. the biomass input in these facilities is mainly based on wood [3].de www.8 t aÀ1 [1]. Karlsruhe.exxonmobil. Esso Raffinerie Ingolstadt.1 Capacities of crude oil conversion of some refineries located in Germany Refinery MiRO Mineraloelraffinerie Oberrhein GmbH & Co. gives rise to substantial amounts of char as byproducts. plus the usually low volumetric energy density.) require technologies that are more complex and more expensive than those employed in conventional petrochemical processes or current direct liquefaction. Fast pyrolysis is described in detail later in this chapter. methanol. for example. especially residual biomass from forestry or agriculture.wrg-whv. straw) is possible. This particular version of a pyrolytic process.92 Diesel from Biomass Table 5. As a result.g. It has been calculated that only large biomass-to-liquid (BtL) plants with synfuel production capacities of at least 1 000 000 t aÀ1 (input at least 6 000 000 t aÀ1 dry lignocellulose) would profit from meaningful economies of scale [2].2 Direct Liquefaction At the time of writing. Germany a Capacity (t aÀ1)a 15 000 000 13 700 000 12 000 000 5 000 000 Source of information www. an account is presented of the state of global R&D in biomass liquefaction and gasification (status of April 2008). even if the liquid condensate yield reaches 60 %.

Although this would be the most efficient way to produce fuel. 15 % water. such as char or coke [5]. without the intermediate steps of gasification and synthesis. as it makes the heating value of HTU-derived oils relatively high (ca. Table 5.02–5 16–19 Hydrothermal biocrude <0.2 Properties of pyrolysis and hydrothermal ‘oil’ from Kroger [12] ¨ Property Water content (%) Density (kg mÀ3) Viscosity (Pas at 20  C) Higher heating value (MJ kgÀ1) Pyrolysis oil 15–30 $1140–1300 0. organic waste or sewage sludge in particular were successfully converted by HTU into approximately 50 % oil (‘biocrude’). Catalytic direct liquefaction has been the subject of a recent review [4]. The direct beneficial consequence of water acting as a reactant and a solvent is that hydrothermal liquefaction produces no solids. while the exergy efficiency is estimated to exceed 50 % [7]. such as acids and sugars.75–1 [16] 30–36 [17] . Biomass is very reactive in water: the polar bonds of the biomass components are attacked by the polar water molecules. The Netherlands. Wood chips.Biomass Liquefaction and Gasification 93 hydrothermal and catalytic liquefaction. ‘Hydrothermal’ refers to conversion in water. see Table 5. and 5 % dissolved organic substances. is always lower in HTU-biocrude than in pyrolysis oils. Moreover. The thermal efficiency of the HTU process ranges between 70 and 90 %. Den Haag. hemicellulose and cellulose are hydrolyzed very quickly. as a consequence. This is an important criterion. the degradation products of biomass are highly soluble in hot compressed water. it is hoped that these processes could achieve higher liquid fuel yields.2. while the biomass employed typically has an oxygen content of approximately 40–50 %. The method of hydrothermal liquefaction investigated best to date is ‘hydrothermal upgrading’ (HTU). with its high natural water content.2 [15] $820–845 [15] 0.2). 5. The water contained in the biomass is an important reactant in the degradation of the polymer structure of biomass. 30–35 MJ kgÀ1. the resulting organic material starts flowing only above 80  C.1).1 Hydrothermal Liquefaction Most biomass feedstocks available are ‘wet’ – that is.1. and is not considered in detail here. and the phenols are more dominant (Figure 5. The word ‘oil’ as used in this context may be misleading: usually. biomass is converted into gaseous products and oil within 5–15 min [6]. without drying. their water contents range up to 95 %. The content of polar compounds.2. Hydrothermal processes have been designed to convert biomass. unfortunately no direct liquefaction process has yet been devised that will deliver a liquid quality necessary for automobile fuel. which are extraordinarily aggressive at elevated temperatures and pressures. At 300–350  C and 15–20 MPa. a process originally developed by Royal Dutch Shell plc. 30 % gas ( > 90 % CO2). The oil so produced contains only approximately 10–15 % oxygen. which suppresses any problems of polymerization. Direct liquefaction refers to the conversion of biomass to fuel by means of special catalysts. usually at elevated temperatures and pressures. while hydrothermal treatment is covered briefly in Section 5.

PyB: Zhang [9]. hemicellulose.1 Classes of compounds found in pyrolysis oils (PyA and PyB) and HTU biocrude (HTA and HTB). to increase the oil yield and to reduce the amount of organic compounds left in the water. HTA and HTB: Karag€ z [10. namely cellulose.2 Slow Pyrolysis Three main classes of organic materials are present in lignocellulose biomass. called Sludge-to-Oil Reactor System (STORS). In addition. The Netherlands. In most cases.14]. hydrogen carbonates or carbonates are used as catalysts [10]. In addition.2. A similar process.13. The high viscosity of HTU-biocrude may be a disadvantage in some applications (e. is used to convert sewage sludge into oil and coke. These two reactions removing oxygen lead to compounds of lower oxygen content [5. The process was demonstrated on a large scale in the hydrothermal upgrading pilot plant of 120 kg hÀ1 capacity successfully operated by TNO-MEP in Apeldoorn. Typically.94 Diesel from Biomass Figure 5. The difference in composition shown in Figure 5. alkali hydroxides. in small engines). The large amount of effluent water containing residual organic compounds is a challenge to the further development of hydrothermal liquefaction.g. 5. 20 % in the coke. have been found to increase the oil yield [18].11]. and 20 % in the aqueous solution [19]. diagram taken from Kr€ ger [12] o o The high phenol content makes HTU-biocrude an interesting feedstock for the production of resins.1 can be ascribed perhaps to the special properties of hot compressed water promoting the elimination of water and carbon dioxide. Some examples of potential European and American . a large project has started to produce a hydrogenated and deoxygenated fuel called HTU-diesel. Numerous studies with catalysts have been conducted to solve this problem. Data sources: PyA: average data from Bridgwater [8]. some 44 % of the initial carbon is retrieved in the oil. such as Co3O4. and lignin.. heterogeneous catalysts. 16 % in the gas. as feeding is only possible at elevated temperatures. The low concentrations of aldehydes and acids lead to a less corrosive behavior of HTU-biocrude compared to pyrolysis oil.

’ which also results from conventional charcoal production. The Reichert Retort Process of Evonik Industries. At temperatures above 600  C. ‘tar. secondary cracking reactions become increasingly significant.1 Conventional Charcoal Production Coarse pieces of wood are gradually heated to about 400  C for many hours by slow partial combustion. hydrogen > 400  C . has a low heat content and can be used as a liquid additive. aqueous condensate. condensable tar vapors decrease.’ which has a relatively high energy content and thus can be used in diesel engines.2. formic acid. formic acid. ash: $2 %. Charcoal has many uses. Minor or special components such as ash. The resultant products are charcoal (C: $80 %. Traditionally. charcoal is produced in kilns with charcoal as the sole product. tar compounds CO.4 Charcoal formation Temperature range <150–200 C <270  C <400  C  Reaction heat Endothermic Weakly endothermic [26] Weakly exothermic [27] Highly exothermic 880 kJ kgÀ1 wood [26] 1200–1600 kJ kgÀ1 wood [27] Weakly endothermic Volatile products Water (little CO2. for example in the bioliq process. methanol. Germany (then Degussa GmbH) and the SIFIC Process (Lambiotte et Cie. whereas conventional industrial charcoal production processes use large retorts of capacities of 100 m3 and more. it may be used as a smokeless solid fuel. hydrogen. Essen. acetic acid) CO2. acetic acid. and the levels of noncondensable small molecular species rise [20]. for example. the aqueous condensate called ‘Scansmoke. formic acid. This conventional technology is described in detail in this chapter as it generates a liquid byproduct. Brussels.2. methane. or as an ore reductant. Belgium) are the most important Table 5. acetic acid CO. A temperature of at least 400  C is needed for the complete decomposition of these organic polymer structures into monomer and oligomer fragments [20]. In addition. which are combined with refining facilities to treat the volatile products.3 Main classes of biomass composition of five dry lignocellulosic species [22–25]. LHV: $30 MJ kgÀ1).3. organic condensate (‘tar’). 5.Biomass Liquefaction and Gasification 95 Table 5.4). and gas (Table 5. formaldehyde. proteins and sugars are not cited Species Beech wood Spruce wood Wheat straw Bagasse Corn stover Lignin 23 27 17 6 19 Cellulose 41 44 39 33 38 Hemicellulose 35 29 38 23 26 energy sources from agriculture and forestry are listed in Table 5. as pure carbon in chemistry. with preference being given to aromatization [21].

Germany. Germany (formerly Chemviron-Carbon Ltd. and Degussa GmbH). Elemental formulae are empirical. An intrinsic advantage of the SIFIC process is its heat balance: the cold noncondensable pyroligneous gas leaves the condenser and is blown into the bottom of the Lambiotte retort. has a high throughput. This heat exchange heats the gas to the reaction temperature of 580  C. A Reichert facility consists of a network of retorts (up to six) and. while the reaction takes place simultaneously downstream in the center of the retort. depending on the size of the wood logs. . in the SIFIC and Lambiotte processes the retort is filled continuously with wood through an opening at the top. while a part of the noncondensable gas is burnt in order to maintain the offgas temperature above 450  C. no data regarding the condensable liquid byproducts generated in this process have been published. falling into a collection vessel. SIFIC process and lambiotte process [30] These processes are very similar. The Karlsruhe Research Center. etc. symbolizing in each case a phase with almost the given composition [28] variants still in use (Figure 5. Charring occurs when this hot effluent contacts the wooden biomass. batch by batch. operated by proFagus GmbH. therefore. and energy for drying and preheating (2 500 MJ heat and 270 MJ electricity per ton of biomass).96 Diesel from Biomass Figure 5. This plant was designed to produce 30 000 t of charcoal per year. the terms Reichert Retort and DEGUSSA Process are used interchangeably. bio crude oil. a vertical retort is filled with wood. Reichert retort process or DEGUSSA process [30] In this chapter. and subsequently closed. For this. which were then gasified into syngas (see Section 5. and their differences will not be described in detail at this point. The offgases generated are then separated from the tar and subsequently condensed into an acetic acid-bearing phase with a high water content. The logs are first heated by hot offgases (450–550  C). One charcoal plant is located in Bodenfelde. wood tar. Germany. while the tar is used as an impregnating agent and as a binder for briquettes. Methanol (‘pyroligneous spirit’) and acetic acid are separated as marketable basic chemicals. whereas the cooled charcoal produced in this way moves downwards. The biomass input of this process is wooden logs of 30 cm maximum length. However. consumes 50 m3 of water per ton of biomass.3.2 Rough reaction equation of slow pyrolysis of dry lignocellulose biomass.2).2). The purge gas flow is directed upwards. The Reichert Retort Process takes between 12 and 18 h to complete. and the charring area moves downwards with the hot gas. 1800 t of pyroligneous spirit. and provides approximately 5200 t of acetic acid. and 12 000 t of bio-oil (also referred to as pyrolysis oil. The process heat is provided by burning the noncondensable part of the pyroligneous gases. Bodenfelde.) [31]. used pyrolysis liquids from the Reichert Retort Process to produce bio-slurries. where it cools the fresh hot charcoal. hot offgas from a neighboring retort is then injected into the top of the retort. flash carbonization at 10 bar pressure is an innovative process variant with higher charcoal yields [29]. Unlike the Reichert Retort Process. Besides conventional slow pyrolysis.

and 19 % gaseous products. However. and the heat being transferred from the shaft of the screw and from heat carriers. A Lambiotte retort has a capacity of 12 000 t of charcoal per year with these industrial inputs: 2500 MJ heat. Ottawa. ON.3). USA. HHV 24 MJ kgÀ1). The reaction is termed ‘intermediate’ pyrolysis for obvious reasons – it is faster than slow pyrolysis.. A 5 t per day demonstration plant for treating chicken litter was used to demonstrate successfully the validity of the concept. phenols. At 400  C. carrying the volatile vapors into the condensers (Figure 5. Advanced BioRefinery Inc. and 40 m3 water per ton of predried wood (10–15 % moisture) [27]. etc. The heat carriers typically are small balls of various materials of several millimeter diameter. the heating rate is mainly determined by the heat transfer within the biomass. The chief advantages of this approach are its relatively low cost and the many applications it allows.3 s. The basic principle of the Haloclean process is relatively simple. Florence. biomass such as straw or contaminated wood. as yet no firm decision has been made in favor of one of these projects. ROI and ABRI process Both. Canada and Renewable Oil International LLC. 46 % liquid. the plant was modified to fit into a new mobile facility designed to convert wood waste (slash) and other agricultural or forest residues [35]. Although several projects for a commercial Haloclean process realization are currently under evaluation.Biomass Liquefaction and Gasification 97 Figure 5. A single screw rotates in a tubular kiln. 110 MJ electricity. and with a gas residence time in the hot area of only 0. Moreover. higher heating value (HHV) 7 MJ kgÀ1] and an organic phase (8 % pyrolytic lignin. .2. The liquid separates into an aqueous phase [38 % with 50 % water. Pilot experiments on 18 t of straw pellets were performed in a Haloclean reactor designed for 50–100 kg/h feed and located at the Karlsruhe Research Center in Karlsruhe.3 Refining of the volatile pyrolysis products from the Reichert Retort Process and then preheats and dries the unpyrolyzed wood in the upper part of the retort. with both the reactor and the reactant being heated from the outside. Germany [34]. have focused on modular small and medium-sized heated augers and pyrolysis plants. The optimum settings for the efficient direct liquefaction of biomass were found to be within the 385–400  C range.. is also referred to as feed material [33]. Although the biomass is heated much faster than in the SIFIC or Reichert Retort Process. 5. pyrolysis typically yielded 35 % char.2.2 Intermediate Pyrolysis by Heated Augers Haloclean The Haloclean process was primarily developed for the thermal treatment of halogenated polymeric wastes (such as flame-retardants in electronic boards) [32]. AL. but slower than fast pyrolysis.

66 62. the liquid yield is only slightly lower than in flash pyrolysis. The optimum temperature range for attaining a high liquid yield is 380–420  C. and a 50 t per day plant for slash will probably be commissioned later in 2008. 5. 5. 60 % bio-oil. 7.5 Fuel properties of ROI pyrolysis bio-oil Fuel properties Wood HHV (MJ kgÀ1) Density (kg mÀ3) Kinematic viscosity at 50  C H2O (wt %) Solids (wt %) Organic C. balances and products (some of the details below were published as a previous release on the ROI homepage in 2004) [36].0.3 Fast Pyrolysis In response to the oil crises of 1973 and 1979. NR ¼ Not reported. needs no inert gas flow. as the mix represents an improved alternative motor fuel that does not tend towards phase separation. the hot vapor is cooled in less than 2 s and. the development of ‘flash’ or ‘fast’ pyrolysis (FP) was initiated mainly in Canada.5. unlike a fluidized bed. 14. H. However. For instance.5 22. 0.6 1100 2. an indirectly heated auger which.5). This type of bio-oil could be blended with bio-ethanol.0. but comparable to the value reported for the Haloclean process [34]. 29.3 7. S (wt %) a Bio-oil from Chicken litter I 4.9 NR NR NR 0. Only limited information has been published concerning the technology.4 cST 21 0. Additional heat may be delivered by an inert material such as steel shot but. The viscosity of bio-oil can be reduced by raising the moisture content of the feedstock.5 44.5. Although ROI and ABRI pursue some activities in this field. This size is currently the maximum for a mobile plant. as a result. the auger from ROI and ABRI is not internally heated. On the other hand.8.2. wet wood is first passed through a dryer and then fed to the reactor. Table 5.98 Diesel from Biomass A commercial 1 t per day plant for the conversion of chicken litter was commissioned in August 2008 in Nova Scotia (NS). O (wt %) Organic N. by incomplete drying.9 Chicken litter II 27.0. as mixing with a coarse heat carrier (such as steel shot) allows only a moderately fast heating.4 70. The ROI and ABRI pyrolysis is not as ‘fast’ as flash pyrolysis. 0. as that country planned to exploit its huge wood .02 HHV ¼ Higher heating value. another consequence is that the bio-oil exhibits a tendency to separate into two phases. 27 % char and 50 % liquid separating into a light phase I (15 %) and a heavy phase II (35 %) [38]. Canada. This can be achieved.6 12. 37. Nevertheless.2 0. and 25 % char.6 NR NR NR 0. bio-oil production from high-nitrogen feedstocks remains unusual and is not typical of biomass liquefaction processes (Table 5. This is significantly lower than that reported in the flash pyrolysis literature [37]. one consequence is that the energy content of the resultant bio-oil decreases.4. chicken litter – an agricultural waste which is difficult to treat – is heated to only 330  C and converted into 23 % gas. 0. 8. for example. Dry wood is converted into 15 % combustible gas. unlike the Haloclean screw.

up to two-thirds of the wood biomass can be converted to a dark brown. as a consequence. pure and homogeneous enough for use as substitutes of heating oil or motor or turbine fuels. Wood bio-oils are stable. The main advantage of FP is its ability to convert solid lignocellulose biomass into a combustible liquid fuel in one single. Formulae are empirical symbolizing a phase of almost the given composition [28] reserves to alleviate some effects of the petroleum shock. was found to be in the range of 475–525  C. The condensates typically comprise a multitude of components and.4). Despite much worldwide development effort over 30 years. Table 5. A list of related references was provided by Yaman [39]. for residence time 0.2. at least for stationary engines.6 Dependence of bio-oil yield on gas residence time in a fluidized-bed reactor [43] Temperature ( C) 500 525 600 700 Bio-oil yield (%).42] (Table 5. Moreover.4 Rough reaction equation of the fast pyrolysis reaction of dry lignocellulose biomass.1 Liquid Condensates Many lignocellulose biomass materials are currently being tested as substrates for fast pyrolysis. Some biomass constituents promote secondary tar cracking and. The optimum temperature for attaining reproducibly a high yield of pyrolysis liquids. Briefly.3. which results in a high condensate yield of up to 75 %. decrease condensate yield and increase char yield [41. especially in industrialized countries. tend to undergo phase separation. especially when these oil substitutes are produced from biomass feedstock.g. Overall. 5. steel shot. small lignocellulose particles of less than 3 mm diameter and with a moisture content below 20 % are mixed quickly with an excess of a grainy heat carrier (e. simple step. Pyrolysis liquid yield also depends significantly on the vapor residence time [40].Biomass Liquefaction and Gasification 99 Figure 5.8 s 75 62 57 33 . viscous ‘bio-oil’ or ‘bio-crude. the ambitious market initially imagined for bio-oils has not yet materialized. sand. and the poor properties and insufficient quality of multicomponent bio-oil mixtures. with high ash contents. Heating and vapor condensation occur in only one or two seconds. etc.. for residence time 0.6).2 s 81 76 75 70 Bio-oil yield (%). such as straw. unfortunately.’ Bio-oil is characterized by a high density of approximately 1200 kg mÀ3 and a LHV of up to 24 MJ kgÀ1. Among the reasons for this sluggish market development are high biomass prices.) at approximately 500  C (Figure 5. as well as on the inorganic components of biomass. tar yields and tar characteristics were seen to vary within a narrow range under these conditions.

8–2.7 0.9 Compound 2-Hydroxy-1-methyl-1cyclopentenene-3-one 2-Methyl-2-cyclopentenone 2-Furaldehyde N-Butyric acid Syringaldeyde Guaiacol 4-Methylguaiacol Eugenol Syringol Vanillin Isoeugenol (cis þ trans) Levoglucosan Cellobiosan Percentage 0.5 % are shown [44] Compound Formaldehyde Acetaldehyde Glycolic acid Glyoxal Methanol (5H)-Furan-2-one Water Hydroxyaldehyde 5-Hydroxymethylfurfural Acetic acid Butanol Propionic acid Hydroxypropanone Percentage 0.5 0. causing them to separate when mixed with conventional hydrocarbon fuels. esters.100 Diesel from Biomass Table 5.3–0.1–0.6 0.2 0.2 Deoxygenation of Bio-Oils Pyrolysis liquids with lower oxygen contents are desirable because of their higher energy contents. the amount of a low-energy phase of the bio-oil rich in water should increase accordingly.1 0.1–0.2–1. Emulsification with hydrocarbons.7 0. carbonyls and their phenolic analogues amounted to 36–40 % of the mix.1–0.8). methanol is used as the standard solvent for pyrolysis tars derived from biomass. the residual 1 % being almost completely soluble as a dark brown solution when dissolved in tetrahydrofuran.0 0.1–1.4–0.6 16–30 2.2–1. Ethanol can also be used.5 0. Only components representing fractions of more than 0.6 0. Moreover. This directly results in an increase in the amount of reaction water. Horne et al.2–0.8 3.5 0. experiments of catalytic deoxygenation .5–0. from a theoretical point of view.5–6.3 Their high oxygen content (up to a maximum of 45 %) make pyrolysis condensates hydrophilic. Approximately 99 % of pyrolysis tar is soluble in methanol.3–0.3–4.47].2 % aliphatics.8–3.8–1. as is demonstrated by gas analyses from noncondensable gases (Table 5. 5.8 0. although such attempts have met with only limited success in the laboratory [46. On the other hand.3. Carcinogenic and mutagenic polycyclic hydrocarbons (PAHs) were also found.2 0. such as ethers. In our laboratory.0–1. oxygen can be removed from organic tar by providing excess hydrogen during the reaction.1–3.5–2.2–0. such as light fuel oil.7).9–1. while their lower polarity means a better compatibility with hydrocarbon fuels.2. characterized bio-oils by chemical fractionation of dewatered pyrolysis oils derived from a mixture of waste wood. In theory.0–4.7 1.8 2.6–1.5 % aromatic hydrocarbons.7 Composition of fast pyrolysis bio-oil.5 0. whereas neutral oxygenated compounds. and polar compounds (such as alcohols and organic acids) to 55–58 % [20] (Table 5.0–2.5 0.3–1. but at concentrations below 120 ppm.4 0.0 0. but requires larger amounts. is one way of increasing the heat content and decreasing the corrosive property of bio-oil [44].5 0.8 0.2 0. Part of the oxygen present in biomass is removed as CO and CO2. as water cannot be separated easily and efficiently from the diverse mix of liquid products. and 0. The concentration ranges shown reflect measurements of four different bio-oils derived from different processes and different feedstocks. They observed the presence of less than 0.3–0.

in West Lorne and in Guelph. the gas residence time is 1–2 s Compound H2S CO2 CO H2 CH4 C2H6 C2H4 Volume (%) 0.2. Canada [54]. while iron traces from the reactor vessels were also destructive.55–0.07–0.23–0.e. have been reported. but only partially to alcohols. while the oxygen concentration was lowered from 44 % to 17–26 %.1 0. These two FP reactors. 5.39 0. although Brazilian bagasse was also used. although pumps and pipings must be adapted so as to overcome its more pronounced corrosive nature.. whereas carbonyls were not reduced to hydrocarbons. The fuel pumps must be . The oil produced from these plants is single-phase oil that is stable for more than six months. hydrotreatment remains a significant cost factor [51] and. by using a sulfided CoMo or NiMo [52] catalyst or with ruthenium catalysts [53].00–0. the carbon content increased from 48 % to 65–74 % in the organic phase. which are bubbling fluidized sand beds (BFB). poisoning of the catalyst was observed: 40 ppm sulfur was sufficient for a significant deactivation. the incomplete hydrogenation of bio-oils under medium conditions was recently examined.3. Canada. i. inert gas (nitrogen) is not included.03–0.03 0. heavy heating oil). A fixed-bed ruthenium catalysis at 150  C and a H2 pressure of 133–140 bar resulted in a preferred hydrogenation of unsaturated or aromatic carbon.8 Gas analysis of noncondensable gases from biomass pyrolysis in a twin-screw reactor (FZK). There is a proportional correlation between the oxygen content of the organic phase and the carbon content of the aqueous phase.0 1. as a result. Ranges are indicated in volumetric percentages.3 Reactor Types Bubbling fluidized beds Two commercial FP plants with capacities of 100 and 200 t of wood residues per day were designed and built by the Dynamotive company.3–50.10 0. fuel #2 (C10–C20 distillate.Biomass Liquefaction and Gasification 101 Table 5. Regardless of the approach. heating oil) or fuel #6 (C20–C70 distillate.85 0. a H-ZSM5 zeolite catalyst [49]. Ontario. When Dynamotive pyrolysis oil from white wood was deoxygenated (31–70 %). Simultaneously.9–9. the higher viscosity of the bio-oil produced in the Dynamotive process.5–1. given the low pH (2–3 for biomass with low nitrogen. the one-phase pyrolysis oil input is converted to a two-phase hydrogenation product due to a decrease in the hydrophilic character of the complex mixture and the higher water content.3 4. Moreover.14–0. or a colloidal FeS catalyst [50].5–54.13 32.9).5–4.1 34.7 Compound C2H2 C3H8 C3H6 Total C4H10 C4H8 C4H6 Total C5Hx Volume (%) 0.19 in a hot vapor stream of biomass pyrolysis. for example with a NiMo catalyst [48]. Vancouver. Unfortunately. necessitates further adaptions [55] (Table 5. The practical use of this bio-oil as a renewable fuel was demonstrated in oil-fired units designed to burn either diesel oil. compared to conventional fuel.7–1. low protein content).06–0. for example. are mostly fed with bark-free Canadian softwood to attain a 68 % bio-oil yield.1 0. Very little retrofitting is necessary for the use of such bio-oil.09 0.26 0.08–0.

The bio-oil is converted into natural special chemicals and products in downstream refining steps. the process is characterized by a very high liquid yield (75 % bio-oil.10). <0. uses circulating fluidized-bed reactors (CFBs) with an overall capacity of 110 t per day of dry wood. the hot section of the turbine was also modified. for on-line cleaning. On the other hand. The efficiency of a diesel engine powered by Ensyn bio-oil has been reported as 43 % on an LHV basis. and 12 % combustible gas.9 Fuel properties of Dynamotive flash pyrolysis bio-oil [54. 13 % char. O. Rika Ltd. Pyrolysis . char is not an undesired byproduct of the Dynamotive flash pyrolysis process. rather.5 1190 75 cST 58 23 <0. The turbine atomizer unit was adapted by adding a preheater step in order to reduce bio-oil viscosity. based on wood feedstock of 8 % moisture) [59]. The char and gas are burned to generate process heat.4 1200 57 cST Not given 21 <0.. the biomass is pressed onto a hot moving disc ( > 500  C) where pyrolysis takes place and the biomass ‘melts’ (Table 5.02 Bio-oil from bagasse 15.7. 7. The reaction time. in Finland. Besides its use for combustion. 44. this bio-oil was demonstrated to be a valuable fuel also for powering a commercial 2. a 0. and a 0. S (%) À1 Bio-oil from pine/spruce 16. patented technology. it is considered a beneficial additive that increases the heating value of the bio-oil (‘biooilplus’ or ‘BOP’). H (%) N. the bubbling fluidized-bed process is economically competitive at a crude oil price of US$ 70 per barrel [56]. or is blended with fossil fuels.65 t hÀ1 pilot plant at the ENEL power station of Bastardo (CFB Ensyn-technology) in Italy. 0. This particular process is called Rapid Thermal Processing (RTP). 0. whereas the main part of the liquid bio-oil is used in modified turbines and diesel engines for power production.4 capable of handling almost twice the volume of conventional fuels because of the lower energy content of bio-oil. Consequently. Circulating fluidized beds Another Canadian company.35 t hÀ1 Wellmann plant with BFB technology in the UK. from heating the biomass up to quenching the pyrolysis products.102 Diesel from Biomass Table 5. there are no commercial biomass FP plants. According to Dynamotive. 13–16 <0.02 Not given Commercial distillate 42–43 820–860 3–6 cST 74 $0.58] Fuel properties LHV (MJ kg ) Density (kg mÀ3) Viscosity at 20  C Flash point ( C) H2O (%) Ash (%) C.5 MW gas turbine (OGT2500).01 84–87. obtained a license on this technology in 2006 for its implementation in Latvia and Ukraine.3 0. a combined-cycle system with a stationary turbine attains approximately 40 %.02 <0. but a number of pilot plants have been abandoned for either technical or economic reasons: Union Fenosa (CFB Ensyntechnology) in Spain. The Latvian biodiesel company. where bio-oil production is planned on a large part of its leased 250 000 ha of farmland [57].5 t hÀ1 Vapo and Fortum plant with proprietary. 0. Ensyn Technologies Inc. Ablative pyrolysis In ablative pyrolysis process.02 42. valuable food flavorings (‘liquid smoke’) are also produced.06. In the EU. typically is less than 2 s. for example.05.

18 56–59. .18. 0. hence. 5–6. hot sand and cold biomass particles of 6 mm maximum size are introduced at the bottom of a rotating cone [65]. built a process demonstration unit for 10 kg hÀ1 of wood slats. 38. <0.4. 6. a cooling unit.7 s.Biomass Liquefaction and Gasification 103 Table 5. and its viscosity was 20 cSt at 20  C and 6 cSt at 50  C. while that of the bio-oil produced was 35 %. Manitowoc. and the reaction zone is only a thin layer a few micrometers wide near the disc surface. during the course of which the sand and biomass are mixed and heat is transferred to the biomass. The bio-oil was preheated to 70  C and burnt in a modified diesel engine of a CHP (120 l hÀ1 bio-oil over a 10 h total running time). thus facilitating the condensation step compared to fluidized beds. Germany. The biomass is not restricted in particle size.9. 0. Ablative pyrolysis has two main advantages: . The vapors generated are sucked in by a low negative differential pressure. It is also worth mentioning that a 6 t per day demonstration unit started operations in 2006 [62].03 Residual bio-oil 16. Enschede. . The hot disc was then heated electrically to 650  C.16 54. after which the reaction gas and vapors passed through a hot cyclone.0 1200 10 cST 24 0. The gas fraction is collected and used to heat the downstream sand.01–0. and an electrostatic filter. The Netherlands. 15 % char. Data from hardwood pyrolysis at Red Arrow Products Company. WI [60] Fuel properties LHV (MJ kg ) Density (kg mÀ3) Kinematic viscosity (60  C) H2O (wt %) Ash (wt %) CHNOS (wt %. and 10 % char [61]. <0. In this case. Centrifugal forces induce an upward movement. whereas the char is removed by mechanical devices. Rotating-cone reactor The rotating-cone reactor was invented at the University of Twente. maf) À1 Whole bio-oil 16. 22 % gas. and 10 % gas. Friction is low in this system.05 is controlled by the heat-transfer rates. In this process. A 100–250 kg hÀ1 plant has been in operation for at least 1 000 h to date. because a thin layer of tar and vapor acts as a lubricant. where the chopped wood feed material was dried by the hot offgases of the burner to 8–10 % moisture.2–0. The PYTEC company in Hamburg.2 1200 27 cST 18–22 0. obtaining yields of 68 % bio-oil. and further developed by BTG Biomass Technology Group. The ablation rate is proportional to the pressure existing between the biomass and the disc. In this process. the length of biomass reacted per unit time. Throughput is determined by the ‘ablation rate’ – that is. the gas residence times in the hot zone were about 1. 20–34. The water content of the wood used in that pilot-scale experiment was 12 %. Typical yields of the ensuing wood pyrolysis are 75 % bio-oil.4. 4 % diesel fuel was mixed with the bio-oil to ensure lubrication [63]. milling efforts are smaller than in other fast pyrolysis processes.10 Fuel properties of Ensyn ‘whole bio-oil’ and ‘residual bio-oil’ (after the extraction of valuable fine chemicals). The Netherlands [64]. Large volumes of inert gases are avoided.

and transport in silo containers. . the quality of the bio-oil generated in this way (25 % water content) could be optimized. The sand is heated in a fluidized bed by burning the char. The hot vapors pass through several cyclones and are quenched downstream by recirculated bio-oil. bioslurries of straw have approximately 10-fold higher mass and energy densities than straw bales. nitrogen. For example. In this way.g. mainly acetic acid. in turn. Without the char. low sulfur. and has an LHV above 10 MJ kgÀ1. bio-oils produced from high-ash lignocellulose (such as straw) have a poor HHV. can be gasified safely. or less. whereas gasification will work with low-purity biooils. improves the handling.4 Bioslurries and Pastes Bioslurries can be considered as low-quality bio-oils mixed with pyrolysis char. Any sludge that can be pumped and atomized pneumatically.. Meanwhile. low-quality. The rotating-cone reactor was scaled up in Malaysia to treat 50 t per day of empty palm fruit bunches [66]. this makes for cheap storage and long-distance transport by rail [67]. such as simplification or cost reduction. Unfortunately. the mechanical complexity is limited. In this latter plant. the advantages of the rotating-cone reactor are comparable to those of the rotating-disc reactor used in ablative pyrolysis.3. the gas flow is low due to an absence of inert gases. Char powder suspension in bio-oils also increases the volumetric energy density which. the energy yield of the bio-oil would be only 65 %. In addition. ash. the palm residues were dried from 65 % to 5–10 % moisture prior to pyrolysis. oxygen. or some other gasification agent. while the residual gas can be used to dry the biomass. homogeneous bio-oil. the bio-oil is routinely fired for start-up purposes of a fluid-bed combustor near Kuala Lumpur. Although the speed of rotation is 300 rpm. Combustion in engines or turbines requires a clean.2. high-ash feedstocks – such as the unused huge surplus cereal straw resources – can now be exploited. 5. When all pulverized pyrolysis char from the FP is mixed into pyrolysis condensates. this is particularly true of pyrophoric pyrolysis char powders. they are also prone to phase separation into a heavy tar phase and an aqueous phase with dissolved oxygenates. biomass liquefaction by pyrolysis produces a highly heterogeneous mix of solid. the slurry or paste will contain up to 90 % of the initial bio-energy. Malaysia [65].104 Diesel from Biomass producing 50 t of bio-oil (2003 data). liquid and gaseous products. 5. and char). a constant flow of bioslurry can easily be pumped into even highly pressurized combustion or gasification chambers for pneumatic atomization with air. which is relatively high. One innovative use of bioslurries is as a fuel for boilers or pressurized entrained flow gasifiers. The resultant experience curve would produce numerous incremental improvements of the FP process. or even ash-containing bio-slurries or pumpable pastes. In this respect. storage. which have a low bulk density.3 Biosynfuels from Biosyngas As discussed in the preceding sections. Also. In the initial plant trials. The refining of bio-crude oil to meet these requirements is not a very promising path. Such low feed quality requirements in gasification constitute a transforming paradigm for achieving a broad market penetration of FP. whereas many applications – such as combustion in engines or turbines – require optimized and highly standardized liquid fuels with low impurities (e.

A number of different products can be synthesized from syngas. Combined with low-temperature fuel cells (FC).11 Examples of production plants via synthesis gas Company Sasol Sasol PetroSA Shell China Energy MHTL BASF a Location Sasolburg. synfuels are named biomass-to-liquid (BtL). South Africa Bintulu. The new mega-methanol plants of Table 5. and gas-to-liquid (GtL). Methanol is produced with more than 99 % selectivity when special Cu-catalysts are employed at 40–80 bar and approximately 250  C [78].3.diesel FT . dimethylether (DME). . Economies of scale were determined empirically to be characterized by a cost degression factor in the range of 0. South Africa Mossel Bay. that is. coal. which were mainly developed during the twentieth century. Belgium Product FT . H2 is generated from syngas in the shift reaction. hydrogen constitutes an important option as an efficient transport fuel. these reactions can be performed with high selectivity when special catalysts are used at certain temperatures and pressures. These syngas processes.e. and many others. between 1910 and 1940.7 [70]. As explained below. are not affected by the origins of syngas (i. South Africa Secunda. 5. Syngas can also be converted to CH4 by means of Ni-catalysts. China Trinidad and Tobago Antwerp.diesel DME Methanol Hydrogena Capacity (t aÀ1) 105 000 [70] 5 280 000 [70] 1 200 000 [71] 640 000 [72] 100 000 [73] 500 000 [74] 100 000 [75] Intermediate in the ammonia production. However. Syngas is derived from fossil fuels by means of Fe. By analogy.diesel FT .diesel FT . CO þ H2O K CO2 þ H2. natural gas or waste). and are now applied commercially on a large industrial scale (Table 5. selective catalytic syngas conversion technologies are well known and available commercially [77]: . alternatively. Malaysia Linyi. they may be referred to generically as XtL. the renewable biomass-derived product fraction in mixed feeds can be determined from its content of radioactive 14C. and downstream synthesis of synthetic biofuels or other basic organic chemicals [68. Fischer–Tropsch (FT) hydrocarbons. . It is assumed that the cost degression curves run exponentially. Shandong. this reaction is used in very large ammonia plants.Biomass Liquefaction and Gasification 105 however. Methane represents another important substitute motor fuel. coal-to-liquid (CtL). CH4 (SNG). biomass.69]. and extensive efforts are being made to optimize biomass conversion into syngas. without discontinuities. including H2. a mixture of CO and H2. . between 100 MW and several GW.. a capacity increase by one order of magnitude corresponds to an increase in capital expenditure by only a factor of 5.11). methanol. Cr-catalysts at approximately 400  C (high-temperature shift) and a subsequent Cucatalyst at approximately 200  C (low-temperature shift).1 Biosyngas Use Downstream synthesis steps based on syngas.

or are presently under construction (Qatar Shell GTL Ltd. This is mostly true when only agricultural residues. BtL development concentrates chiefly on syngas production. Two of these plants are sufficient to feed a large and more economical MtS (Methanol-to-Synfuel) plant attaining a 1 000 000 t aÀ1 hydrocarbon output [80].’ The SASOL company in Johannesburg. Moreover. 5. The main reasons for this approach were the abundance of lignocellulose materials and the fact that waste utilization does not interfere with the production of agricultural crops for human or animal nutrition [84]. Malaysia. on average. such as straw or wood residues. was popularly known as Schwarzheide ‘knock water. to approximately 50 t kmÀ2 in MiddleEurope. in South Africa (today merged with other companies to PetroSA. technology. Together with forest residues. FZK). most methanol-based processes proceed via DME as an intermediate. This alone represents a significant source of lignocellulose material amounting. straight-chain hydrocarbons were developed during the 1920s and applied in Germany during World War II to produce up to 600 000 t aÀ1 of motor synfuel. RSA). Interest in GtL technology has grown over the past two decades. but improved. One 200 000 t aÀ1 facility in Schwarzheide (then East Germany) was in operation as late as 1965. roughly half of the cereal straw harvest is not needed to maintain soil fertility. feedstock preparation differs widely depending on the types and compositions of the feedstock used for syngas generation. The process uses low-quality. a joint venture between Royal Dutch Shell and Qatar Petroleum). The first two commercial GtL facilities were commissioned in 1993 by Royal Dutch Shell plc in Bintuly. This obviously implies a limit on size.3.2 ‘bioliq’ Bioslurry Gasification [81–83] The concept of bioslurry gasification was developed at the Karlsruhe Research Center (Forschungszentrum Karlsruhe. as delivery radii of more than 200 km would be necessary . Qatar recently started production (Oryx GTL. now produces 6 000 000 t aÀ1 of FT synfuel from coal by a similar. What distinguishes these processes are essentially the front-end steps of syngas production. Very large GtL production capacities of several Mt aÀ1 in Ras Laffan. with a low MON of 75. Neat DME is a clean diesel fuel especially for cold climates. and by Mossgas Ltd.106 Diesel from Biomass . the Lurgi company. on average about 200 000 t aÀ1 of residual lignocellulose biomass can thus be made available in those regions within a 30 km radius [67]. As a result. South Africa. forest residues of stem wood harvesting. Parow. Similar plant capacities are considered for DME-production. This synfuel. Germany. in particular. Germany. cheap lignocellulose waste. or residues of the pulp and paper industry are used. Fischer–Tropsch syntheses for long. a joint venture between Sasol and Qatar Petroleum). have a capacity of 1 700 000 t aÀ1 [79]. such as straw. methanol and DME are precursors of a large variety of organic chemicals and fuels. Manufacturing know-how gained by practicing the CtL and GtL processes on various industrial scales is now being transferred to the BtL process.or a two-step process. these chemical conversion routes being identical after the production of clean syngas with the required H2/CO ratio. and thus remains available as an unused surplus. A methanol catalyst supplemented by an alumina or zeolite dehydration catalyst is used in either a one. In rural European areas.

Biomass Liquefaction and Gasification 107 Figure 5. nonwoody biomass residues containing more ash and heteroatoms (e. The reactor used is a twin-screw mixer running at approximately 500  C to produce a condensate yield of 50 % or more. ‘Straw’ is used below as a generic term describing all thin-walled.85] in many regional plants. The technical concept is outlined in Figure 5.g. nitrogen) than wood.5. central further processing (bio-slurry gasification and syngas conversion) in a large central plant to supply a large central biosynfuel plant with a minimum of 6 000 000 t aÀ1 of lignocellulose materials to enable production of at least 1 000 000 t aÀ1 of biosynfuel [2].5 Simplified block flow diagram of the ‘bioliq’ two-step bio-slurry gasification process for synfuel or chemicals production from biomass (byproducts: high-pressure steam and electricity). Local biomass liquefaction by pyrolysis. The pyrolysis liquids and char arising are combined into a dense bioslurry or paste that is transported .8.. fast-growing. The biomass is first liquefied by FP [3.

which result mainly from handling.6) designed by Lurgi. After syngas cleaning and adjustment of the H2/CO ratio to approximately 2 by a combination of the water gas shift reaction and CO2 removal.89]. its design details thus could benefit from experience gained by GtL and CtL plants. which lead to high capital expenses and operating costs. Germany.108 Diesel from Biomass by rail to a large central biosynfuel plant for gasification and synthesis. Frankfurt. For that purpose.3. a 2 MW(th) equivalent of a 500 kg hÀ1 feed material twin-screw FP pilot facility (see Figure 5. It is important to note that the distance of rail transport does not add much to the overall transport costs. the pure syngas is converted catalytically into a range of useful chemical products or chemical building blocks. Consequently. or hydrogen. Sweden. Future Energy. 2004. 5. for four bio-slurry gasification campaigns in 2002. 2003.3 Biosyngas Applications in BIGCC and CHP Demonstration Plants 5.3. A huge central BtL or biosynfuel plant of this type. with a bioslurry throughput of approximately 0. was rented from its successive owners. which is above the ash melting point. while those for methanol or DME synthesis are run at > 50 bar and 70–80 bar. respectively. Unlike designs which employ atmospheric gasification. The most common variants of FT-synthesis typically are run at pressures of 10–40 bar. such as FT-diesel. methanol. Slurries originating from 30 to 50 or more of these regional pyrolysis plants can be shipped by rail over distances of up to 300–500 km to a central facility for syngas generation and use. are avoided in this design through the use of pressurized gasification. a practically tar-free. low-methane syngas was obtained reproducibly at a carbon conversion in excess of 99 %.3. Sustec. Within the bioliq pilot plant.5 t hÀ1 and at 1600–1200  C. methane. Industrial-scale operations showed no major process deviation. Germany. Successful gasification experiments with various bioslurries were conducted in a pressurized entrained flow GSP-type pilot gasifier at 26 bar pressure [88. no major issues are expected to arise in these operations. a pilot gasification facility in Freiberg. would be similar to existing GtL and CtL facilities. While downstream syngas cleaning and synthesis have not been tested to date by FZK. pressurized. The critical steps in syngas generation via bioslurry production and gasification were tested and verified on the 3 MW(th) pilot plant scale [86]. and 2005. was a designed and built by Foster Wheeler and SYCON in 1991–93 and commissioned in . Consistent with thermodynamic equilibrium theory. the energy-consuming intermediate syngas compression steps. with several GW of thermal biomass feed. as they exist commercially on various scales and benefit from the know-how and experience accumulated in GTL and CTL plants. The pilot plant is to define a detailed design enabling the construction of a commercial FP facility with an air-dry biomass throughput of 20–30 t hÀ1. Pyrolysis experiments were conducted at the Karlsruhe Research Center (FZK) with a 10–20 kg hÀ1 process development unit [87]. entrained flow gasifier operated slightly above the pressure of downstream synthesis (30–100 bar) and at temperatures above the ash melting point. is now being commissioned on the site of FZK. The core of bioslurry gasification is a large. feed materials with high ash contents can be treated successfully. BBP (Babcock Borsig Power). dimethylether.1 The V€rnamo Pressurized CFB Gasifier a The first pressurized CFB pilot gasifier for biomass located in V€rnamo.

g. dried. comminuted wood fuel (e. The gas cooler. but closed in 2000 after a highly successful operation as a Biomass Integrated Gasification Combined Cycle (BIGCC) demonstration plant and a CHP gasifier plant. A reliable supply of hot water during nontest periods was guaranteed by a additional grate-fired boilers. Despite all of the initial technical difficulties being overcome.Biomass Liquefaction and Gasification 109 Figure 5.5 t hÀ1 bioliq fast pyrolysis pilot plant at the Karlsruhe Research Center. Much was learned in these pilot-scale experiments regarding gas quality. The aim of the test program was to demonstrate the technical and economical feasibility of the biomass IGCC concept of the Bioflow technology. mÀ3) in the turbine produced 4. Hot syngas carried the bed material up into a cyclone. The feedstocks successfully used in the plant were different wood fuels. and turbine operation. bark. hot gas cleaning.ON Sverige AB). which was of a fire tube design.2 MW of electricity. NJ and Sydkraft. a downstream candle filter (Schumacher.. Combustion of the low calorific gas (5 MJ std. . Sweden (today E. Malmo. while solids returned to the bottom of the gasifier. Commissioning started in February 2008 1993–96. was used to lower the temperature of the effluent to 400  C. the plant failed for economic reasons and was closed in 2000. Germany) was used to remove particulates. wood chips) was forced into a lockhopper and screw-fed into the air-blown CFB gasifier a few meters above the reactor bottom. The capital investment in this demonstration plant was reported to be e25 000 000. In this plant. Crailsheim. The plant was run until 1999. The average gasification temperatures were slightly below 1000  C at 18 bar operating pressure. fuel flexibility. and had a refractory lining. 6 MW(el) were fed into the public grid. plant control. as developed by Foster Wheeler. in August 2007. Clinton. The gasifier was equipped with a cyclone and a return leg. Of the total fuel input of 18 MW. and 9 MW(th) were supplied to the district heating network of V€rnamo. straw and refuse-derived fuels.6 A 0.

An additional boiler is available for heat production only. the feed temperature required for the Jenbacher gas engine to produce electricity and district heat. The separate aircombustion reactor prevents any dilution of syngas by airborne N2. The indirectly heated 8 MW(th) g€ssing CFB biomass gasifier G€ssing. The only solid residue is fly ash from the combustor with a carbon content below 0. As a result. it is proposed that the V€rnamo facility is converted into an education a and training center dedicated to BtL technology development and production. The plant backfitting thus comprised in particular hot-gas cleaning. The fuel capacity of the CHP plant is 8 MW (th). has approximately 3800 inhabitants. mÀ3. and the total plant efficiency regularly exceeds 80 %. corresponding to 50 t per day of wood chips. The design levels of 4.mÀ3. The main feedstock was wooden biomass gasified at 10–20 bar pressure. the energy supply of G€ssing is now u derived completely from renewable energy sources.5 MW(th) district heat and 2 MW(el) electricity generated in GE-Jenbacher internal combustion engines have been met. operating at atmospheric pressure and a temperature of approximately 900  C. A water-cooled heat exchanger reduces the temperature to 150  C. The project started in 2004 and was planned to run for five years. Funds were contributed by many partners. the tar content of the clean product gas is below 20 mg std.5 %. the comminuted wood biomass is mixed with an excess of hot heat carrier at approximately 900  C to generate raw syngas by adding steam. this relatively low carbon content is an important feature of the system. Among the first actions to be taken were a bio-diesel plant and a district heating CHP plant (investment costs of approximately e10 000 000) [91]. where the olivine is reheated in a bubbling fluidized bed by char combustion with air. . after which the particles plus some tar are removed with a fabric filter and recycled into the gasifier. m3 hÀ1 of H2 equivalent. above all. with an expected H2 production of the 18 MW(th) facility in the range of 3500 std. an Austrian town u u situated near the Hungarian border. This project was to achieve pilot-scale production of clean. as it eases ash handling. and technical planning for this policy began in 2003. a o Sweden [90]. the gasifier is fluidized with steam plus syngas. Upgrading raw syngas to clean syngas with an H2/CO-ratio of approximately 2 was the crucial step in process development. this gasifier is run on olivine sand as a heat carrier recycled in a closed loop via a separate combustion reactor. which was a part of the 6th European Union Framework Program and coordinated by V€xj€ University. The rationale for the EU Chrisgas project was that clean H2-rich syngas lends itself particularly well to synthesizing practically any motor fuel. In a nutshell. the CH4 content is high ( > 10 vol %). the V€rnamo facility was revived within the EU Chrisgas project. The plant employs a fast internal circulating fluidized-bed gasifier (FICFB). e8 400 000 from the Swedish Energy Agency. In the gasifier. the G€ssing district decided that its energy requirement should be met u completely from renewable sources. A scrubber subsequently lowers the temperature to 40  C. After little more than a decade of continuous technological improvement. At the end of the last century. catalytic tar removal. and e4 600 000 from industrial project partners. The HHV of the gas is constant at 12 MJstd. and water gas shift. this allothermal gasifier was developed at the Technical University of Vienna. requires a better syngas purification train. The raw syngas is cooled and cleaned in a two-stage system. Upon completion of the Chrisgas project. Retrofitting a BIGCC plant into a biosynfuel production plant. including e9 500 000 from the EU. and the combustor with air. hydrogen-rich syngas from lignocellulose materials.110 Diesel from Biomass However.

20 Æ 5. particularly. flexible spectrum of valuable organic chemicals. The unconverted residue (mainly methane) is available for downstream power or electricity generation by combustion. low-CH4 raw syngas high carbon conversion the possibility to use high gasification pressures the possibility to reach high capacities in excess of 1 GW flexibility in feeds and. However. FTu synthesis and methanization are being investigated in small slip streams. In addition. high-pressure steam. 25 Æ 5. the implications of complex polygeneration should be considered in the context of emerging thermochemical biorefineries. biochemical and physico-chemical biorefineries are part of the future organic chemical industry converting the available multitude of biofeedstocks into a broad.4 Polygeneration Concepts Among the current R&D projects on biosyngas generation and utilization in G€ssing. or electricity. and N2. The G€ssing experiments performed in a raw-syngas slipstream mainly serve to u develop.3. There is no need to adjust the H2/CO-ratio in the CO shift reaction. 5. the poor economic performance of biosynfuel production in industrialized countries remains the major hurdle to commercialization. The range of compositions (in vol %) is: H2. and evaluate suitable polygeneration concepts within the RENEW project of the 5th EU Framework Program. in the choice of fluids and powders. For example. All of the BtL concepts outlined below may serve as the backbones of future thermochemical biorefineries producing chemicals and biosynfuel plus energy (mainly electricity) as secondary products. A GSP-type gasifier (see ‘Schwarze Pumpe’) with a special cooling screen also digests fuels with high ash and salt contents. CO2. .5–1 l hÀ1). tar-free. there is a single-pass synthesis without maximum yield (no CO shift). 40 Æ 5. 4 Æ 1. The application of this process hinges on the technological and economical feasibility of more stringent biomass preparation by converting the raw material – the biomass – into a gas. . and a downstream electricity and steam generation. After thorough raw syngas cleaning. a pumpable slurry or a fine powder that can easily be fed to high-pressure gasification chambers.Biomass Liquefaction and Gasification 111 5. in addition to having a long service life of more than 20 years and allowing fast start-up and shut-down. a liquid. most of the CO and H2 can be converted into a biosynfuel mix in a single pass through a small FT-synthesis reactor (0. for example. Combinations of thermochemical. especially when an Fe-catalyst is used. CH4. the risk of the prices of commodity fossil fuels dropping . 10 Æ 2. and materials in addition to energy in various forms.3. which also catalyzes the shift reaction during FT synthesis. CO.5 Entrained Flow Gasification for Biosynfuel and Chemicals Production The pressurized entrained flow gasification of biomass [92. . such as heat. test. . Polygeneration refers to a variant of syngas use where several products are generated simultaneously with a minimum technical effort. Even at oil prices.93] offers many advantages in syngas generation and downstream synthesis: . The allothermal gasifier generates concentrated syngas undiluted with N2. of more than US$ 100 per barrel. synfuels.

a large BGL fixed-bed gasifier with liquid slag removal has been built and tested at this facility. Germany (i. !1300  C 80 bar. 950  C $80 bar. Bio-slurries from bio-oil plus char Concentrated black liquor from pulp mill Char powder from torrefaction Gasifier conditions GSP-type 130 MW b-plant: 4 bar. the ‘Schwarze Pumpe’ (Black Pump) gasification complex in East Germany (with 36 fixed-bed and two entrained-flow gasifiers) ranked second in the world for coal gasification after the SASOL company based in Secunda.5.12 Major developers of pressurized entrained flow gasifiers for biomass Developer. The fixed-bed gasifiers with a rotating grate have a 3. Windsor. Frankfurt. $30 bar.6 m inner diameter and are operated below the ash melting point (800–1300  C) [95]. slurry. UK. powders from waste and lignite Hot pyrolysis gas from autothermal pyrolysis. owned by Centrica plc. with six fixed-bed gasifiers of 50 MW(th) each and a 130 MW(th) GSP-type entrained-flow gasifier which is run at 26 bar for the treatment of a mix of brown coal and waste [95]. Sweden Energy Centre of the Netherlands.1 The ‘Schwarze Pumpe’ Gasification Complex Some 20 years ago. Recently. thus protecting the reaction chamber from . Germany [96]. in addition to lignite used to balance the heat fluctuation resulting from the diversity of wastes. only the SVZ (‘Sekund€r verwertungszentrum’) owned by Sustec Schwarze a Pumpe GmbH. chemical quench with fine char Fast pyrolysis of lignocellulose. country Schwarze Pumpe. briquettes and many other products derived from brown coal.112 Diesel from Biomass Table 5. eventually.) The feed materials treated in that plant comprise approximately 500 000 t aÀ1 of municipal solid waste and various other wastes. All gasifiers are oxygen-blown and operated at 25 bar. Germany Choren. (BGL stands for British Gas/Lurgi. South Africa.e. A workforce of approximately 20 000 employees supplied East Germany with town gas.12). Following the German reunification in 1990.. The viscous slag is drained down the inner wall of a special cooling screen. !1200  C (pilot plant 26 bar) ca. BtL in developing countries could become competitive there sooner because of the lower biomass and. The Netherlands to lower mean cost levels represents a significant risk to be overcome only if the petroleum and gas markets stabilize at a level that is high enough to ensure economic viability of the new processes. Spreetal. Germany Feed and pretreatment Diverse liquids. lignite for town gas production was substituted stepwise by natural gas. and Lurgi GmbH. the part of the Schwarze Pumpe complex since 1995 dedicated to the secondary use of raw materials) has survived [94]. Today. Germany Chemrec. 1200  C Research Center Karlsruhe. lower technology costs (Table 5.. at 1600–1200  C. 5. The entrained-flow gasifier operates above the ash melting point. Despite the lack of an infrastructure.3. as the gasifier was developed jointly by British Gas.

5. The raw syngas. saturated with moisture at 220  C. using the synthesis technology from Haldor Topsoe A/S. the SVZ abruptly suspended its gasification activities due to the uneconomic German refuse market. In their concept of ‘Black Liquor Gasification to Automotive Fuels’ (BLGAF). The commercialization of this special technology. DME. while a highly selective copper catalyst with a recycle loop is employed to convert the 55 000 std. which comprises a special entrained-flow gasifier operation. The facilities comprise an efficient Rectisol unit at À60  C using methanol as solvent. concentrated black liquor from the evaporator is fed to a 30 bar O2blown black liquor gasifier for conversion to raw syngas and recycling the cooking chemicals that result from pulping (green liquor). or approximately 9 000 000 m3 per year of motor fuels. approximately 60 TWh of fuel can be produced from 90 TWh of low-grade biomass. On the mid-term a reactivation of the gasification facilities is planned with regards to methanol production from lignite powder [97].3. during the same time.3. In the gas cleaning unit the tars.5 % of the total energy consumption of the EU. The 45 MW(el) gas turbine consumes 50 000 std. Furthermore.5. each modified pulp mill in the EU (ca. mÀ3. the tar removed from the raw syngas of the fixed-bed gasifiers is gasified in the entrained-flow gasifier. The process was first tested in a 1 MW(th) atmospheric pressure entrained-flow gasifier in . In this latter process. which is located in Freiberg. Unconverted syngas can be recycled and partly purged to remove any inert gas. sulfur and CO2 are removed.3 CHOREN Carbo-V Process The CHOREN company. 60 plants) can produce at least 100 000 t of motor fuel. is cooled and scrubbed so as to generate steam at medium and low pressures. hence. Thus.2 CHEMREC Black Liquor Gasification Technology Chemrec AB is a Swedish company with headquarters in Stockholm. developed the Carbo-V process [100–102].5. m3 hÀ1 of syngas with an LHV of 14 Æ 2 MJ std. the BLGAF process does not replace the old recovery boilers and the bark boiler with new combustors. The production of 4–5 t DME per day is planned by spring 2010. a consortium with Chemrec and others started the BioDME project. or methanol by black liquor gasification [98]. biomass is added to a pulp mill and the residue is converted into motor fuel. Denmark. despite its limited capacity. the electricity generated in this process totals 75 MW(el). Germany. m3 hÀ1 of syngas at 40 bar and 250  C to 120 000 t aÀ1 of methanol. The entrained-flow gasifier accommodates gas. As a result. Sweden. The clean adjusted syngas is compressed from 30 to 60 bar and routed to the DME or a methanol synthesis loop. In September 2008. Interestingly.Biomass Liquefaction and Gasification 113 corrosion. and the H2/CO-ratio is adjusted to 2 by the shift reaction. liquid. but rather with gasifiers and steam turbines. supported by the EU and the Swedish Energy Agency [99]. a typical pulp mill produces 400 000 t of dry pulp. This represents about 2. and the DME/methanol is purified in a final distillation unit. Lyngby. a feasibility study for a plant producing 70 000 t of DME each year is being elaborated. 5. the equivalent of 20 000 000 t aÀ1 of CO2 emission reduction. The pulp and paper industry employs approximately five million workers in the EU. At the end of 2007. powder and slurry feed. Each year. is almost certain because of its lower cost compared to other BtL processes. and produces 120 TWh of black liquor which at present is burnt in Tomlinson boilers to recover the cooking chemicals.

This industrial technique generates ‘roasted wood. In addition. it thus has the potential to make significant reductions in the logistics burden of very large plants.’ which still contains approximately 90 % of the initial bioenergy. where wooden raw materials are not only available in large quantities but are also accessible by ship. This contrasts with areas such as the Baltic. and act as an important enabler for operating very large and economically competitive central biosynfuel/chemicals plants. where they are subsequently gasified with pure oxygen above the ash melting point at 1300–1500  C. at Petten. slightly hydrophilic brittle wood resulting from this treatment is milled at reasonable industrial effort to a powder that is sufficiently fine for direct feeding with a pressurized inert carrier gas. torrefaction destroys the cellulose fibers. Torrefaction can be defined as the slow pyrolysis of lignocellulose biomass at relatively low temperatures of 250–300  C. Royal Dutch Shell plc provides the know-how for downstream FT-synthesis. is currently elucidating the technical details of this concept [93]. the process is conducted as follows. 5. In the ‘b-plant. A 40 MW(th) pilot ‘b-facility’ for gasifier operation at 4 bar is now under construction in Freiberg. pulverized and blown into the hot gasifier discharge flow from the gasifier chamber to cool the gas to approximately 900  C by the endothermic carbon gasification reaction. and the ‘torrefied’ blackish brown. several tons of biosynfuel have been produced so far in downstream test facilities. the design capacity of which is 15 000 t of biosynfuel each year. after which the comminuted dried biomass is pyrolyzed by partial combustion at 400–500  C in a specially developed cylindrical reactor equipped with mixer paddles. The Netherlands. The technical effort and industrial inputs necessary for the direct milling of dry lignocellulose materials to such a small size would be great. These physical characteristics are critical to achieving complete conversion within 1 s of residence time in a hot entrained-flow gasifier flame above 1000  C. Waste heat from the process is first used to dry the biomass to a moisture content of 15–20 %. The hot pyrolysis gases and vapors are routed directly into the gasifier.5. Following a multistep washing and cleaning process. the pure syngas is fed to the synthesis unit. On the other hand. to an atmospheric or pressurized entrained-flow gasifier. Biomass transport is a critical issue in the Netherlands. After this ‘chemical quenching’ step. For example.114 Diesel from Biomass the so-called ‘a-facility’ in Freiberg. or milled to a powder for use as a fertilizer. which requires the use of tightly locked hoppers. Briefly. the remaining char powder is recovered and recycled to the gasifier burner.3. offering woodland resources in excess of 200 000 000 ha. The FT-synthesis line is supplied by Royal Dutch Shell plc and is based on the Shell SMDS-process [103].’ the pyrolysis and gasification are run at 4 bar pressure. it is worth noting that the higher pressure used in this process requires an intermediate gas compression. the ECN company. given the high shear resistance of cellulose fibers.1 mm in diameter.4 BtL by Gasification of ‘Torrefied’ Wood A solid feed for an entrained-flow gasifier must be a fine powder of particles less than 0. This material is in a form that is suitable for storage and transport. The pyrolytic char is cooled. which is a relatively small country with a very limited forest area of only 300 000 ha. The vitrified solid slag from the gasifier can either be granulated for road building. .

an optimum H2/CO-ratio must be set with the CO shift reaction. and approximately 80 % of the energy can be recovered. is exothermal with ÀDH. However. Large biomass-fired IGCC stations or cofiring in large coal or natural gas-fired IGCC power stations. in most cases. and Cl heteroatoms by hydrogenation. mainly in the form of liquid fuels for use in engines or turbines. 5. raw syngas from wood gasification can be used after little or moderate purification as an ash-free. the oil constituents are separated by distillation. to fuel not only trucks but also passenger cars with dry pieces of wood a few centimeters in size. A large 100 000 km2 collection area of forest and agricultural residues (45 % of the straw harvest) results in approximately 6 000 000 t per year of dry lignocellulose input and. . Depending on the product and the catalyst. not be too different from that of a modern oil refinery of approximately 10 000 000 t per year. biomass gasification for the heat. The corresponding BtL facilities can thus benefit from the design and operating experience with XtL-systems of comparable size. The capacity of a biosynfuel plant should. Gas motors or turbines for the generation of power or electricity (after dust and tar removal). The heat of reaction of . gasification. O. On the other hand. synthesis. and is more efficient and flexible. Fuel gas for small or medium-sized CHP plants. the conversion of lignocellulose biomass to biosynfuel in a BtL process is achieved via a sequence of chemical transformations. Some 7 % of the energy content of the oil is consumed in the refinery processes in the combustion of poor-quality products. The trend therefore is in favor of larger units for gasification applications. and final product workup is economically viable only in sufficiently large plants. on the other hand. Each of these chemical reactions must be exergonic to proceed with ÀDG and.Biomass Liquefaction and Gasification 115 5. . in approximately 1 000 000 t per year of biosynfuel output. smokeless fuel gas for special combustion applications where direct biomass combustion is impractical: . High-temperature process heat. N. N. Except for the fluid catalytic cracker (FCC) and the removal of small amounts of S. . followed by CO2 removal. therefore. for example. for example in cement or lime kilns. syngas cleaning. and other catalyst poisons. The whole complex process chain of feedstock preparation. it is more complex and more expensive. namely the more than one million small wood gasifiers built in Europe during and shortly after World War II.3. There is a historic exception to this. power and electricity markets entails less pollution than direct biomass combustion. ultimately.7 Energy Efficiency In a conventional petroleum refinery.6 Various Syngas Applications and Economies of Scale [104] Small-scale wood combustion for domestic heating remains the dominant bioenergy application. It should be noted that synthesis of fuels or organic chemicals from raw syngas requires syngas to be carefully cleaned of all S. down to the 10 ppbrange [105]. Cl. On the one hand.3. very little chemical conversion is involved.

a logistical term). 5. none of the direct processes of biomass liquefaction is ready for the market. via the gasification and heterogeneous catalytic reactions of synthesis gas. and amounts to 10–20 % of the initial bioenergy. In theory. biosynfuels could become economically competitive at a lower level. In developing countries. The heats of reaction of various versions of FT syntheses amount to 20–25 % of syngas energy. where biomass is cheaper. As BtL energy efficiency is twofold lower. in 2007. e1. .e. ca. Neglecting feedstock costs.8 Comparison of Oil-Derived Motor Fuel and Biosynfuel Manufacturing Costs Approximately 7 t of air-dry (15 % moisture) or 6 t of dry lignocellulose input is needed to produce 1 t of biosynfuel. biosynfuel manufacturing costs are roughly one order of magnitude higher than those of conventional petroleum refineries. Although catalytic . a BtL plant could be designed which would be self-sufficient in terms of energy [67]. and might well rise in the future as supply and demand go out of balance. . due mainly to the higher complexity of BtL conversion technology.116 Diesel from Biomass gasification appears as sensible heat of the hot raw syngas. the remaining 40–50 % of bioenergy should be recoverable for electricity generation or the production of highpressure steam. As a result. e65 per barrel. Typical energy yields in a welldesigned BtL process are 40–50 % for FT hydrocarbons or methanol. A large share can be used for O2-production in the cryogenic air separation unit. extending well into a range of approximately e500 tÀ1 [2]. in 2007 the costs of biosynfuels were still above those of oil-derived transport fuels.4 Perspective The liquefaction of biomass can be divided into direct and indirect processes.3. it must be emphasized that biosynfuels would become economically competitive if crude oil prices were to reach a level of US$ 150 per barrel. or a maximum of US$ 111 per barrel on the spot markets). .b. then the greater will be the technical effort and the lower the energy yield in the final product.5 kWhÀ1 for wood or straw f. A typical BtL plant compares to a typical conventional oil refinery as follows [2]: . although with typical FT-synthesis temperatures of only 200  C this energy cannot be used efficiently. or approximately e60 tÀ1 for air-dried lignocellulosic materials). 5. To date. the crude oil and biomass costs to produce 1 t of motor fuel are comparable if: (i) the oil prices are in the same range of e500 tÀ1 (i. The more exothermal reactions that are combined in successive process steps. the development costs of biomass would be difficult to predict.. However. However.o. depending on O2 consumption. and thus are negligible. and (ii) bioenergy in its raw material form costs half the price of oil (in 2007. As thermal insulation losses amount to only a few percent. (free on board. a lower energy efficiency (factor of 2) a higher mass input (factor of 5) a higher technical process complexity (factor of 2) a significantly smaller plant capacity compared to a crude refinery (at least by a factor of 10).

these fuels are far from the quality standards of transportation fuels. so as to increase energetic efficiency. substitute natural gas (SNG) and hydrogen are all well known and standardized. fuels..g. improved heat recovery. and the Chemrec plant (currently under construction) are also too small in terms of economy. Experiments to improve HTU and pyrolysis liquids by cleaning and grading up (e. Whilst successful tests of fuels generated in this have been conducted in turbines and boilers. methanol. the results of pilot experiments are eagerly be awaited. in contrast to the . some promising results have been reported during the past few years. a permanent and long-term operation was neither planned nor realized due to its too-small size and. for an eco-economic assessment. (ii) gasification/syngas cleaning and conditioning. The syntheses of hydrocarbons. conditioning and synthesis steps. accordingly. The main hurdles of liquefaction via pyrolysis and hydrothermal processes are the quality issues of the product. and it is likely that optimization and standardization will lead to the potential application of these fuels in heavy-duty engines.Biomass Liquefaction and Gasification 117 liquefaction is a relatively young branch of BtL research. However. Whilst olefins. the biochemical conversion of syngas to ethanol is currently being examined [108. The charm of direct liquefaction lies in its expected simplicity and.109]. for example by high-pressure gasification. by hydrogenation) are currently in progress. while catalytic liquefaction still remains to be a subject of controversy and has not become established within the scientific community [4]. and other oxygenated fuels are also feasible. and (iii) heterogeneous catalytic synthesis into the desired fuel and possible byproducts. because the synthesis reactions of the BtL trains resemble those of the GtL and CtL-processes. ethanol. Moreover. or high-pressure–high-temperature gas cleaning. The previously operated a-plant of Choren. biomass feeding. poor economy. however. Unless direct liquefaction is actually not an appropriate choice for the production of transportation fuels. it may be used favorably for electricity or heat generation and. the Fischer–Tropsch facility in G€ssing was used exclusively for research purposes. An economic comparison to other plants is difficult. further development is also required for the syngas production. and the conversion processes under consideration of multifeed ability. supplying both electricity and district heat. for the production of organic chemicals. because the majority of the funding of these investigations has been via subsidies for R&D. the future trends of the automotive industry will not lead to simpler fuels. for example to meet more restrictive emission standards or for use in ambitious combined-cycle combustion engines that currently are under development [106]. Although the 18 MW(th) CFB gasifier in V€rnamo a functioned well during its demonstration period. the research progress has not yet exceeded bench-scale [107]. With regards to the state of development. u it was better designed and more suitable for a long-term operation. The indirect liquefaction of biomass requires a complex multistep procedure that essentially includes: (i) biomass pretreatment. dimethylether. Yet. Yet. no commercially operated liquid fuel trains or even gasifiers are operated today. as a result. and was much too small to u draw economic conclusions. in its higher energy efficiency and better economies compared to multistep processes. Whilst the G€ssing 8 MW(th) gasifier was even smaller. The major research demand in biomass liquefaction via the gasification route mainly concerns the front-end processes of biomass preparation and preconversion. but rather to even more demanding fuels. and the use of heterogeneous and changing feed stock quality and high ash contents. For example. to a less extent. However.

E. Bridgwater. 2.C. Since. the main hurdle would be to buildup a large network of 30 to 40 regional pyrolysis plants. 6. F. 45. A. Supercrit. 3. N. An example of this can be seen in the actual and recent investments in Chinese CtL plants. and could operate economically if BtL fuels were to enjoy tax privileges.J.V. Swaan. B. and to develop reliable cost estimates. 2007. According to the plans of Choren. Chem. . Henrich. E. BP Statistical Review of World Energy June 2008.118 Diesel from Biomass V€rnamo and G€ssing systems. 2513–2518. In our estimation. A. K. 2006. Here. Y. they have been designed specifically for liquid fuel a u production (FT-diesel and DME.Reaktionsmechanismen und Produktverteilungen report no. 2729. 4. biomass for BtL fuels competes with biomass for food and materials production. Dinjus. commercialization appears to be irrelevant. in the long term a mixture of diverse renewable and fossil energy sources appears unavoidable. Available at: www. Sci. Dahmen. 7. 1990. it is essential that an appropriate logistic and business model is developed. that may be implemented into an existing paper mill complex. 805.R. G. Kruse. 1. Peters. which have indicated a renaissance in coal gasification and its synthetic chemical products. E. it is essential that these processes are not only codeveloped but also integrated for future use. their plant behind the b-plant in commission now will be the gammaplant planned for Lubmin. 114-50-10-0337/05-B. all of these plants are well suited for acquiring experience of operational data.J. Berlin. only 20 % at best of the primary energy needed in 2100 will be covered by biomass. albeit at a lower capacity. but in future the intention is to employ coal gasification.. in principle the Schwarze Pumpe gasifier could also digest biomass. In the past. a 5. what about the other 80 %? It appears that coal. So. Sustain. municipal and industrial waste was gasified. to allow for considerations of scale-up. C. in applying a de-centralized/centralized concept. 2002. the existing cost estimates suggest that a poly-generation plant should have a capacity of at least 1 GW. in other words. ecologically friendly fuel. 7–11 May 2007. with a zero purchase price and transportation costs. Behrendt. 4. Germany. the opportunity persists of combined BtL and CtL plants. Schulz-T€nnies. Neubauer. however. Fluid Phase Equilibria. 44. A Chemrec plant could produce a black liquor residue on an economic basis. N. Zhong. Thus. C. BP Distribution Services. although in both the public domain and in politics a stringent BtL fuel can today be more easily described as a ‘green’ or. respectively). oil sands and other lower-grade fossil feed stocks will become increasingly important as crude oil production declines. Today. With an annual maximum output of approximately 1000 t. Fluids. Wilmes. Zobel. Proceedings 15th European Biomass Conference. J. 362. as only then would a large gasifier and synthesis complex be capable of working at full capacity. 194–197. 2000. Since XtL processes differ only in their front-end processes. Dinjus. Technische Univerit€t Berlin. Peferoen. Eng. The competitiveness of methanol synthesis in the Schwarze Pumpe depended heavily on world methanol market prices and the German refuse market. For the bioliq process.V. References 1. D. Goudriaan. Peacocke. Renew. Germany. London. F. Direktverfl€ssigung von o u Biomasse . UK. However. 39. Germany. Energy Rev.bp. that has been designed for an annual FT-diesel output of 200 000 t.

A. 37. 75. Springer. Fransham. Ferrer. 2891. Winnacker/Kuechler. Kruse. 1051. 17. .A. Ledesma. H. 16. 2007. 25.J.E. J. van Estrik. Euopean Patent Specification EP1354172B1. Kruse.. 2004. 2001. Dittmeyer. R. 494. Srokol. Williams. P. Sci. 32. X. Muto. Energie aus Biomasse. in R. R.J. Karag€z. Germany. Stockholm. S.B. Health B. 41. 11. A. 31 August p. 18. A. Band 5: Organische Zwischenverbindungen. Naber. UK.Chemischen Prozesses zur Verwertung von Abf€llen a aus Elektro. Mochidzuki. M. Hornung. J. V. 4th edn. A. 2004. CPL Press. Fransham. 2005. 26. 30. A. in H. Handbook of Charcoal Making. HTUÒ -DIESEL FROM BIOMASS.nrbp. J. 2006. Harnisch. Chemisch-technische Verwertung von Biomasse. Hajaligol.. Germany. et al. Koch. Sun. homepage version in May 2004. Wiley-VCH. Bockhorn. Chemische Technologie Vol. 1996..die ‘Haloclean’-Pyrolyse. 2007.J. A. Brocksiepe. K. Strik.F. B. 642. R. Bouche. G. A. Hallen.B. Meier. The Netherlands 2002. Canadian patent CA 2351892. Manag. and report FZKA 7301 of Forschungszentrum Karlsruhe. 11–18. The Netherlands. o 11. Naber. Available at: http://www. Kaltschmitt (ed. Aiello. 23. P. Anal. et al. Russell. A. 28. J. 62. et al.. J. Steiner. 97. 22. 36. 471–475. Energy Conv. A. Bhaskar. 33.V. p. Weinheim. 5th ed. www. Chang.K. Apfelbacher. Y.. S. Baker.S. 90. Bhaskar. H. W. Kr€ger. 42.M.presentation at SYNBIOS conference. Chem. W. Paredes. 1984. E. A. 339. 31. 19. SGINC1-07. Tomkinson.J. 23. 2002.. 3. 35. W. Nelson. Polymere.S. 57. Sealock. Z. Muto. Res. T.ecotraffic. Keim.und Elektronikkaltger€ten . D.Biomass Liquefaction and Gasification 119 8.J. Y. 21. 87. Pohl. Supercrit. A. M.G. 6247-02-01-11-1002. Diploma thesis. 14th European Biomass Conference. Eng. 2007. Chem. Ind. Watanabe. Q. Germany. M. D. Chemische Technik: Prozesse und Produkte. 1985.G. W. 2. L. Res. AL (www. 1999.T. Elliott. 5: Organische Technologie I. PhD thesis. Reichel Publishing Company. University of a Stuttgart. Y. ACS Division of Fuel Chemistry Conference. 2002.A. Molton. Winnacker (eds).M. Schweers. Lee. 2007. 29. 20. Fast Pyrolysis of Biomass. Heemskerk. 2006. T. 84. 1996. 2002. Goudriaan. L. SD. Silva. Germany. Application of direct thermal liquefaction for the conversion of cellulosic biomass. Environ. Res. T. Antal. Dinjus. Wang.. 2005. P. Ullmann’s Encyclopedia of Industrial Chemistry.A. 8. Bayer. o 12. Facey. Develop. J.. vol. Sun.I. 42. Bioresource Tech. M. Carbohydrate Res. Carl Hanser Verlag. Boe. 913. Karlsruhe. D. M. F. Prod. L. A.G. Zhang.. Biores.renewableoil. Leipzig. report no. 109. A. T. Waymack. Available at www. Sweden.). Schnitzer. Sakata..pdf. W.A. Fuel. Horne. South Dakota State University. 2005. Wiley-VCH. u p. Weinheim. 875. Fassbender. Baird. 1981. T. J. Goudriaan. P. Kreysa. Owens. 9. o 13.Composition of Herbaceous Biomass Feedstocks. Berlin.. 2005. Jerenyama. 38.E. 14. Hornung. 1. 27. 35.C. 341. University of Applied Science. Bridgwater. 2003. 10. Ind. 71. France.T. E.renewableoilinternational. F.M.. vol. Fluids. Oberholz (eds). Sakata. A. Xu. Germany. Maschmeyer. P. biofuel B. 18–20 May 2005. M€nchen. 361. Entwicklung eines Thermisch . G. K. R. Pyrol. Report No. D.C. C. 48. E. Kaminsky. 15. W. V. Chem. 3690.G. Florence. Emrich. 34. Henrich. Carbohydrate Res.N. 1230. Eng.. M. 47–57. 24. Germany. Paris. 2007. F. J. vol. Ind. Eng.. Karag€z. 2007. 83. Tech. 331. P. Fuel. Monreal. A. Peters. USA. et al. C. 1717. Fisher. 1996. Washington DC. Appl. Presentation from Renewable Oil InternationalÒ LLC. J.A. 2007. Transportbrandstoffen via hydrothermale liquificatie van biomassa met het. Polymer Degradation and Stability.

J. 45.J. Chem. London. World Renewable Energy Congress. 1536. 63. van Swaaij. D. 657. o Newbury. Velen. D. R. Den Haag. 68. F. Klaubert. 42.P. Linyi. G. Tech. Yaman. 251. Germany. 1994.E. Shandong. Sanford. 1997. Graham. 293. Capart. S. in A. 176. Leible.D..J. in A.V. Res. 19. Synthesis gas from Biomass for Fuels and Chemicals. Available online at: Wagenaar. Neuenschwander. 1715. 227. E. . 72. Blackie Academic and Scientific.H. Citiroglu.btgworld. Megaritis. B. 2004. 58.chinaenergy. Di Blasi.. Berns.B. in A. in A.C. Anal. 36. Radlein. C.J. UK. 2007. 69. 1985. 2006. UK. University of Waterloo. A. 30. Chem. 2. Florijn. p. W. 46. Kandiyoti. Sch€ll.R. 2001. J. Developments in Thermochemical Biomass UK.G. S. Samolada. Gansekoele. Appl.A. 52. Herod. Information from: www. 60. Lafferty. C.. www. 1994. Hu. van der Drift. Solana. Newbury. J. 55.. D. 2006. 73. L.” Renewable Energy World Magazine. K. 24. A. UK. Eng. 2002.. J. Fuel Proc. Biomass Bioenergy. Cao. Boerrigter. R. 31 January 2006. Biomass Bioenergy. 5 December 2002. CPL A. Freel. Meier. 2006.V. H. May/June 2007.M. 1998. Snape. Blackie Academic & Professional. 2005. 61.renewableenergyworld. Proceedings. 54. Energy.W. Aberdeen. Sturzl. Pyrol. in A. 2006.C.B. Bridgwater. Hart.G. 611. 2. H. Ikura. 5891.V. Buhler. Hogan.V..The commercial co-firing of RTP bio-oil at the manitowoc public utilities power generation station. 77. D. 44.A. Gercel. Today. C. Markgraf. Sugar J. J. Liden.V. W. 2003.). Barynin.M. “Dynamotive to make bio-oil in Ukraine and Baltic States. Luengo. 45. 49. UK. Scott. Klaubert. Boocock (eds). p. 2007.E. S. Advances in Thermochemical Biomass Conversion. M.E. Knoef. 65. I. P. 7 August. 1372. Prins. 2007. 259. E. status from March UK. Proceedings. Sch€ll. Chemical Engineering. Branca. Fuel. D. 1: Perspectives on Biomass gasification. Morris.. The Netherlands. M. 51. Elliott. M. G. H. 221. p. Snape. 5109. J. 1047. H. 62. 67. F. Donald. S. 7. Blackie Academic & Professional. M. Huffman. R. MASc Thesis. 1999. Babu. et al. Science in Thermal and Chemical Biomass Conversion. B. 64. 48. D. Sch€ll. Lange. Workshop No. L. Markgraf.. T.M. A. 51. Biofuels Journal. Berks. D. Dry. Khezami.. FZKA 7170. Appl. R.G.V. C. 4. J. R. D. China. Pindoria. Feinman.N. March 2008. Ekinci. 1527.G. BG. 66. 41. A. p. D. 103. Rocha.G. Energy Conversion Manag. Canadin Business Magazine. O. CPL Press. Bridgwater (ed. Catal. 115. Majerski. D. ThermalNet. et al. Shell GTL plant in Bintulu.E. Newbury. et al. Elliott. Berks. o 2006.C. Meier. Chapman & Hall. S. Boocock (eds). record 188 (on CD). Advances in Thermochemical Biomass Conversion. Bridgwater. UK. Int. R. 2001. Putun. Homepage of Royal Dutch Shell plc. J. M. Jones. S.gastechnology.). J. Boocock (eds). 2008. D. 69. 59. 70.V. 1994. Snape. 1993. M. Fagbemi. R. 1016. 53. 651. 40. 2008. Org.C.. G. Homepage from China Energy. p. 49. Sci. L. Assink. Wissenschaftliche Berichte. Neuenschwander. 71. C. 2007. Science in Thermal and Chemical Biomass Conversion. London. C. Vasalos. 1994. Stanciulescu. p. Klaubert. 1997. J. January 2006. 43. 1999. CPL Scientific Publishing Service Ltd. p. C. ECH-C06-001. 56. 299. London. 45.A. Forschungszena trum Karlsruhe. Bridgwater (ed. Piskorz. IEA bioenergy task 33. The Catalyst Review Newsletter. www.A. K€lber. Kappler. Available online at: www.G.B. 23. Eng. 590.120 Diesel from Biomass 39. Malaysia.P. G. Bridgwater (ed. 57. Meier. Faix. D. A. D.pyne. Development in Thermochemical Biomass Conversion. 50. IEA bioenergy task 33. Int. Available at: www. J..F. 71.) Success & Visions for o Bioenergy. Available at: www. Bridgwater. 47. DGMK Meeting. Pyne-Newsletter. Geochem. London.

100. 99. et al. 95. E. 93. P. Pyrolysis and Gasification of Biomass and Waste. E. Proceedings. www. 2000. 2004. Rudloff.A. Beyond Oil and Gas: The Methanol Economy. Higman. K. 189. April 2008. Available online at: www. M. 1875. Dahmen. K. 2004. Steinhardt. W. 2001. Germany. Amsterdam. T.A. 813. Bridgwater. Proceedings. 3. 2004. Umweltbundesamt. 2003. Intermediate Report. H. Wender. p. 76. 5th ed. 7th Holzenergiesymposion. Weinheim. 94. 227–239. 2002. Anorganische Stickstoffverbindungen. Erd€l Erdgas Kohle. Wolf.K. Henrich. van Hoek. Trinidad and Tobago. Schingnitz. pp. 2008. W. Timmerhaus. R. Germany. Wiley-VCH. Bridgwater (eds). 243. Zweibett-Wirbelschichtvergasung in G€ssing (A) mit 2 MWel/4. 17–22 June 2002. 5th ed. Raffelt. 98. Mag.. 83. F. Seifert. Lindblom. Davies. www. p. W. Switzerland. NY. Group/GasNet 2005. B€hland. 10–14 May 2004. M. Ripperger. R. A. www. Prakash. 90. Proceedings. 1588.methanex. 101. 2008. Betriebserfahrungen und Wirtschaftlichkeit. 86. Techn. Ahlvik.. Dinjus. Rome. 2003. T.S.5 MWth. Trinidad and Tobago. Bengtsson.svz-gmbh. W. S. Plant Design and Economics for Chemical Engineers. 31–34. Sweden.G. Gail. Schneider. 2007. K€gel. Henrich. P. Energy & Fuels. Langanke. BWK. Lacey. Henrich.V. 1. Seifert. I. 84. Berglin. Buttker. J. G. Weinheim. A. 92. Germany. New York. UK 2003. E. 18. M. 48. P.V. Ekbom. 2006. B. Czernik. 1. 54. R. N. 96. 590. 78. Chemische Technik. Zukunftsmarkt Synthetische Biokraftstoffe. 248. 247. Vierrath. UK 2006. 10–12. 80. 2003. Oil Gas Europ. Bridgwater. E. 10–14 May 2004. F. H. Velen. VDI-Berichte. Germany. 75. Dessau. R. Homepage from Methanol Holdings (Trinidad) Limited. Oberholz (eds). in R. Gasification. 88. 2005. www. D.H. USA. Volkmann.html. Fuel Proc. 85. 2004. Henrich. BTG Biomass Tech. Kreysa. Handbook of Biomass Gasification.J. S. J. CA. DGMK meeting. Newbury. Science in Thermal and Chemical Biomass Conversion. Dinjus. 97. C. 2008. DGMK Meeting. Henrich. 10. Homepage from Methanex Trinidad Limited. A. E. Technical and Commercial Feasibility Study of Black Liquor Gasification with Methanol/DME Production as Motor Fuels for Automotive Uses. Schingnitz.B. Weirich. Rome. 169. 2002.V. K. Velen. R. Wolf. Henrich. E. Dittmeyer. Hofbauer. G. McGraw-Hill. T. H. Dinjus. A. M. vol. 228–232. p. M.S. 628. Liebner. 298. B. E. B.E. Elsevier Science. Nachwachsende Rohstoffe. o Schalke. 776.M.. u Germany. Konzept. Spreetal. A. G. Chemie Ingenieur Technik. H. 511–526. Dinjus. USA. Boocock (eds). Report under the framework of the EU . Anorganische Grundstoffe. Keim.D. o 81. West (eds). 102. Herbert. 12th EU Conference on Biomass. Homepage of sustec Schwarze Pumpe F. Wagner. Thompson.S. Proceedings. W. Peters. 2007. Reaction of synthesis gas. J. CPL Scientific Press. Proceedings. vol. IT. Buttker.E. Wasserstoff aus Biomasse. Landwirtschaftsverlag GmbH.. Velen. Prozesse und Produkte. M. D. 89. sect. 29. Germany.Biomass Liquefaction and Gasification 121 74. in A.programme B. Zurich.ttmethanol. M€nster. Weirich. 2nd World Conference on Biomass for Energy. Homepage of the EU project BioDME. CPL Scientific Press. Proceedings. NL. B. S. 103. 1565. 82. pp. Goeppert. Kulzer. Wilfinger. . Stockholm. Stahl. H. 74. 79. E. Scott. Germany. u 91. Newbury. E. van der Burgt. p. Knoef. Clean Hydrogen-rich Synthesis Gas (Chrisgas). Olah. E. 13. Weirich. Rauch.chrisgas. Zwischenprodukte. B.K. Proceedings of the Conference on Coal Gasification Systems and Synthetic Fuels for Power Generation. M. 57. 1996. G. 1985. J. 2004. 87. Gos. 77. pp. N. Germany. Stahl. 18 October 2002. E. DGMK meeting. San Francisco. o 2nd World Conference on Biomass for Energy. Wiley-VCH Verlag. 2008.

R.J. 86 (5).P. The Netherlands.N. Tijmensen. Spivey.M. 2006. D. Nannen. Hamelinck.Perspektiven f€r Biokraftstoffe der 2. Guo. van Kasteren. van Hardeveld.R.. Review of applications of gases from biomass gasification. ECN. R. ECN-RX06-066.P. 2006. W. B. J. Biomass and Bioenergy. Rauch.A.L. 1514–1528.N. Faaij. 129–152. Huhnke.Bio-ethanol from Syngas. Datar.R.C. Lewis. R. 106. 2004. Cateni. Dizdarevic. H. A. 2002. Technisch Universiteit Eindhoven. Biotech. 2007. Chem. . 105. J. Rev.M. Bioeng. C. Report number 0268040420012. M.G. Generation. Verberne. H. R. Eindhoven. 587–594. 23. A. 108. Petten. M.M. R. van der Waall. R. 2005. Shenkman.. Germany. Giessen. The Netherlands. Egbebi. J. u Presentation at the VDL-Fachtagung “Bioenergie”. 107.S.J.Kraftstoffe aus Biomasse . 36.122 Diesel from Biomass 104. Soc. Boerrigter. 109.

especially in European countries. a carbon-neutral fuel with low emissions and a high internal combustion efficiency. Consequently. with the number of diesel engines being predicted to increase to 1. crude oil-based diesel.1 billion by the year 2020 [1]. Jian Xu and Yong Yang 6. as compared to conventional diesel fuels [2]. Fischer–Tropsch diesel (FTD). Nasib Qureshi. Biomass to Biofuels: Strategies for Global Industries and Hideaki Yukawa Ó 2010 John Wiley & Sons.75% of the current petroleum diesel with biomass transportation fuels by the end of 2010. has been proven to be effective in dramatically reducing the emission of sulfur dioxide. The gasification/reforming into syngas of carbon-containing materials such as coal. automobile manufacturers worldwide are increasingly viewing FTD as a feasible alternative diesel engine fuel. industrially. usually consists of three steps: . Fischer–Tropsch diesel can be obtained from syngas via a Fischer–Tropsch synthesis (F-T synthesis) which. or sustainable biomass. and probably by more than 15% by the year 2020 [3]. As an alternative fuel to the conventional. namely a high fuel efficiency and a low impact on the existing distribution infrastructure. natural gas. dictate the need for a ‘super-clean’ diesel – that is. Hans Blaschek e .1 Introduction Diesel-powered heavy duty trucks and more efficient diesel cars have been widely used in the industrialized nations. it is particularly worth noting that the European Commission’s target is to replace 5.6 Diesel from Syngas Yong-Wang Li. Ltd Edited by Alain Verts. which has a high cetane number and almost zero sulfur content. given its two primary differentiating attributes. Increasingly stringent environmental regulations. nitrogen oxides and particulate matter. From a practical viewpoint. however.

6. followed by a discussion of recent variations in F-T syntheses. although small amounts of isomers and oxygenates are also produced in addition to the primary byproducts. a full picture is obtained of diesel production via F-T synthesis from a typical feedstock such as biomass. it is increasingly likely that biomass will come to represent a primary source in the energy mix of most countries. 6]. leading to the Anderson–Schulz–Flory (ASF) distribution: logðWn =nÞ ¼ n log a þ constant ð6:3Þ In this equation.2 Overview of Fischer–Tropsch Synthesis The F-T synthesis converts syngas into hydrocarbons (mainly alkane and alkene) through catalytic hydrogenation [4]. The chain length distribution of the hydrocarbons thus produced has been modeled by the stepwise carbon chain growth mechanism on the catalyst surface. With ever-increasing concerns over global warming and peak oil consumption/prices. This chapter provides a brief review of the development of the F-T synthesis process. A higher a-value means a higher selectivity of heavy hydrocarbons. water and secondary CO2 [5.124 . the ÀCH2À groups represent chain-type hydrocarbons ranging from methane to heavier waxes. CTL (for coal-to-liquids). These reactions are. namely GTL (for gas-to-liquids). Wn represents the mass fraction of the species with carbon number n. when there is a need to produce high yields of heavy hydrocarbons that can in turn be upgraded into diesel fuels [4. which describes the production of syngas from biomass. the process can. By combining this information with that in Chapter 5. thus providing another option to both consumers and car manufacturers. The manufacturing operations implemented in typical F-T processes are then analyzed from an economics point of view.9. whereas a is defined as the chain growth factor. Moreover. this change will occur to a great extent via F-T syntheses that will enable the manufacture of additional alternative fuels. depending on the feedstock used to generate the syngas. Diesel from Biomass A catalytic F-T synthesis. . be expressed by the following chemical reactions in their simplified forms: The hydrocarbon formation reaction: 2H2 þ CO ! ÀCH2 À þ H2 O The water-gas shift (WGS): CO þ H2 O ! H2 þ CO2 ð6:2Þ ð6:1Þ In these equations. in principle. The hydrocarbons produced from F-T synthesis are mainly straight-chain alkanes and alkenes. 5]. . Fischer–Tropsch syntheses may be described differently. and BTL (for biomass-to-liquids). or biomass-based carboncontaining materials into several of the useful hydrocarbons that have served as the backbone of modern motor fuels and the feedstock of chemical plants. in principle. A typical F-T synthesis for diesel production is preferentially performed at an a-value greater than 0. which usually involves a mild hydrocracking process. natural gas. that is. A product work-up. very important due to their applications in converting syngas derived from coal.

and Ru – can effectively catalyze F-T synthesis reactions. a total manufacturing capacity of more than 200 000 tons per year of F-T synthesis-derived products was reached [5. Of these metals. Apart from the above-described main active metals. for practical applications. the ruthenium nanocluster catalyst can display excellent activity and selectivity towards liquid oils. has – from a practical point of view – not proceeded much beyond those initial discoveries.and cobalt-based catalysts can be considered as practical F-T catalysts. these catalysts are composed of metals. a theoretical yield of 208. Driven by war and industry demands. F-T synthesis technology in Germany quickly evolved into a scaled-up technology. Ludwigshafen. ruthenium is the most active. that have been reported from Beijing University (patent owned by Synfuels China: CN 200710099011 PCT/CN2008/000886) [12]. Moreover. however. and patented the catalyst in 1925 [5]. Hence. Co. remains to be determined. These findings suggest that. at about the time when ammonia synthesis catalysts were discovered by Haber and Bosch [8. the concomitant occurrence of WGS reactions over the same catalyst surface further complicates the process [7. The F-T synthesis reactions can occur with industrially meaningful rates only by the promotion of catalysts at elevated temperature. Notably. Ni. 15]. 14. on the basis of the syngas fed into the F-T synthesis loop. Sabtier and Senderens observed that methane could be formed over Co.3 g of hydrocarbon can be derived from each cubic meter of syngas converted. and this results in the formation of an array of hydrocarbon products. using ruthenium nanocluster catalysts. it becomes clear that only iron. Fe. it is worth highlighting the recent developments in aqueous-phase F-T synthesis. Consequently. 13]. In a practical F-T synthesis process. It is perhaps surprising that the open-domain knowledge of F-T catalyst formulation. and Ni metal catalysts via CO hydrogenation [7]. In other words. The company Badische Anilin. Typically. whilst the aqueous phase may destroy conventional F-T catalysts [9]. 14]. Germany (BASF) then reported the production of liquids over a cobalt catalyst in 1913 [7. Fundamentally. despite having been explored to great depths over the past 80 years following the first studies in Germany [9–11]. Fischer and Tropsch were the first to report hydrocarbon formation over alkalized iron in 1923. in 1936. . the understanding is that four Group VIII metals – Fe. Whether the opportunity exists to formulate a novel F-T catalyst by using nanotechnology. but its high cost and low availability rules it out for large-scale applications.3 Historical Development of the Fischer–Tropsch Synthesis Process Fischer–Tropsch synthesis catalysts were first developed in Germany during the early 1900s. 6. many more aspects of F-T catalysis must be carefully considered to develop a successful industrial F-T catalyst. the yields of the desired products are lower than this value by a factor of 10–20%. Nickel is also very active. The major complexity of F-T synthesis in chemistry and catalysis is due to the fact that multiple reaction paths occur on the catalyst surface. 8].und Soda-Fabrik. In 1902. this subject has been reviewed comprehensively [9]. but produces much more methane during F-T synthesis than do the other metals.Diesel from Syngas 125 The stoichiometric relationship of the above chemical reactions define that 3 moles of syngas are transformed into 14 g of hydrocarbon products. such as iron or cobalt [4–6].

Sasol decided to construct two larger coal-based F-T synthesis plants in addition to Sasol I [17. which was commissioned with a 34 000 barrels per day (bbl dÀ1) capacity in 2006 in Sasol’s Qatar GTL project (see Table 6. MTG process and ZSM-5 catalyst Sasol II and III commissioned R&D on slurry phase reactor started Improved Synthol Test slurry-bed reactor Sasol I slurry bed reactor commissioned Shell GTL Sasol Advanced Synthol (SAS) Sasol Qatar GTL project commissioned Country Germany Germany USA South Africa South Africa US South Africa South Africa South Africa South Africa South Africa Malaysia South Africa Qatar . respectively.1. and later to South Africa [5. Further details of the earlier developments of F-T synthesis technology are available in reviews [5. gradually shifted from Germany to the United States. 17]. this development was critical as it enabled the production of gasoline at high yields from CO and H2 [17–19]. 20. the Arge fixed-bed reactor that initially was developed in Germany [5. the fluidized-bed reactor concept was developed in the US. 23–25] Time 1923 1938 1945–1950 1950 1955 1976 1980/82 1980 1983–89 1990 1993 1993 1995 2006– Process technology events Fischer and Tropsch discovered hydrocarbon formation over alkalized iron Nine F-T plants constructed Development of fluidized-bed F-T synthesis Sasol founded Sasol I commissioned Mobil slurry-bed reactor. 7. In parallel.1 F-T synthesis development: 80 years of history [9–12. 17. 18] was successfully commissioned in 1955 on a commercial scale. Among the salient developments achieved by this company is an improved version of the fluidized-bed reactor and the lowtemperature slurry phase reactor. 17. much of the F-T synthesis knowledge. Their 12 000 bbl dÀ1 commercial-scale plant has been successfully running since 1993. 14. It is noteworthy that in the field of GTL. iron and cobalt catalysts can be used in the latter reactor. Qatar) to invest about US$ 5 billion to build a plant with a capacity of 140 000 bbl dÀ1GTL products. The combined capacity of the three Sasol plants was then about 6 million tons per year. 9–12. The first phase of the project is due to become operational between Table 6. 17. The plant was named Sasol I by the newly founded Sasol Ltd company (originally South African Coal and Oil). Together with the Kellogg entrained-bed reactor (which later developed into the Sasol Synthol reactor [5. 18. 15. 17. 16. 18]. These two plants were commissioned in 1980 and 1982. as well as further developments. A timeline of F-T synthesis process development is presented in Table 6. at the site now known as Sasolburg. 22]. 15. Both. 21]). the company Royal Dutch Shell plc (Shell) pursued from the start a different F-T synthesis process route that is based on a tailored cobalt catalyst used in fixed-bed reactors. Moreover. 17]. from which it can be seen that Sasol has invested a significant amount of resources in F-T synthesis process development since the 1980s. Shell recently partnered with Qatar Petroleum (Doha.1). During the oil crisis of the 1970s.126 Diesel from Biomass After World War II. in South Africa [5. 14. primarily naphtha and transport fuels.

The second phase is due to be commissioned two years later [23]. 3000 h 10 000 ton aÀ1. (ii) pilot-scale slurry-phase F-T synthesis processes. 33] Time 1937 Technology events Cobalt-based catalyst. which used a precipitated iron catalyst [also known as Table 6. the fixedbed F-T synthesis technology. Jinzhou. 1953 1980s 1993–94 1996–96 1997–1999 2000–04 2003–04 2005–2009 Fluidized-bed pilotscale test in Dalian ICC’s Two-stage fixedbed reactor (MFT) Industrial pilot test Single tube test ICC-IIA. including the economy of energy-thirsty countries such as China or India. 4500 ton aÀ1. As a result. while the slurry-phase F-T synthesis was investigated only at the laboratory scale. China has actively been participating in the R&D of F-T synthesis processes to harness the potential value of its hundreds of billion tons of economically mineable coal and huge amounts of biomass resources.Diesel from Syngas 127 2008 and 2009. iron catalyst 100 ton aÀ1 pilot test 2000 ton aÀ1 3000 h test Slurry and kinetics 1000 ton aÀ1. Among the research themes being actively pursued are the development of: (i) highly active and low-cost F-T synthesis catalysts. 4706 h 160 000 ton aÀ1 Site Jinzhou 1952–1962: owned by No. 6 Petroleum Plant.2 F-T synthesis in China: a brief history [25. After the war in 1952–1962 in operation with total oil production of 400 000 ton. first version of German technology Details Japan military plan for a F-T plant of 30000 t aÀ1 capacity. B Slurry-bed reactor pilot test Yankuang pilot plant Synfuels China demonstration plants Dalian Petroleum Research Institute Shanxi Dai County Fertilizer Plant Shanxi Jincheng Fertilizer Plant ICC ICC ICC Lunan Fertilizer Plant Yitai Coal Group (Inner Mongolia) Luan Coal Group (Luan) . and (iii) related process integration technologies. producing 70 000 bbl dÀ1 of GTL products. the Institute of Coal Chemistry (ICC) (Shanxi.2. but never in operation during the war. In 1993. emphasis was placed on research aimed at optimizing the iron catalyst used in fixed-bed reactors. China) is playing an important role in optimizing F-T synthesis technology under the framework of the China Coal Utilization Program. essentially forcing them to seek practical alternative means of securing appropriate energy supplies. the F-T synthesis development program in China can be divided into two different stages. Before 1997. As shown in Table 6. In particular. The jump in crude oil price that occurred shortly after entering the new millennium has greatly impacted the worldwide economy. The cyclic and regional nature of the interest in the F-T synthesis technology is positively correlated to both the periodic fluctuation in the price of crude oil [8] and to the energy mix and infrastructure of any specific country.

two iron catalysts were tested in laboratory-scale reactors with detailed kinetics investigations [26–32]. will be expected to pave the way to further scale-up to a truly commercial-scale plant. From this diagram. 21].128 Diesel from Biomass modified Fischer–Tropsch (MFT) technology] was scaled up to the pilot plant scale (ca. purified syngas must have a (H2 þ CO) volumetric content above 98 %. which usually dissolves the F-T synthesis oxygenates. will be commissioned between 2008 and 2009 [33]. and this led to major improvements in several key technologies that made the process work. two demonstration CTL plants. In 2000. 6. or sent to a tailgas treatment unit to recover light condensate from the fuel gas. A pilot plant with a capacity of 15–20 bbl dÀ1 for the slurry-phase F-T synthesis process development was commissioned from 2001 to 2004. the unconverted syngas from the F-T synthesis reactor is either recycled for further conversion.4. naphtha. Currently. and liquid petroleum gas (LPG). The raw syngas is subsequently subjected to a purification process to remove contaminants in order to maintain the sulfur concentration below 50 ppb. each with a capacity of 160 000 tons per year (ca.4. The light condensate recovered is then combined with the major wax and condensate stream from the F-T synthesis reactor. especially in the field of process engineering. irrespective of the syngas source. Depending on the actual process integration arrangement. the suspended three-phase fluid-bed (slurry bed) F-T synthesis reactor was . 3500 bbl dÀ1). 40 bbl dÀ1) [25]. a dedicated water treatment unit in the F-T synthesis process is required. Likewise.2 F-T Synthesis Reactors The initial version of industrial F-T synthesis reactors was the fixed-bed format. and subsequently sent to the product processing unit for upgrading into end products such as diesel. The acid gas stream with high concentration of H2S and other sulfur compounds exiting from the gas purification unit is sent to a sulfur recovery unit to collect elemental sulfur. the syngas thus generated is fed to the F-T synthesis reactor to produce hydrocarbons (gaseous. Since a significant amount of reaction water is generated. The experience gained from these demonstration projects. 18. part of the inlet raw syngas stream from the gasification unit is first shifted in a WGS reactor to adjust the H2/CO ratio to approximately 1. In order to meet quality specifications.1 Modern Fischer–Tropsch Synthesis Processes Overview of F-T Synthesis Process Technologies A simplified flow chart of the Fischer–Tropsch process is shown in Figure 6.2. liquid.0. A brief timeline of F-T process development history in China is provided in Table 6. and wax) and water.1.6–2. the ICC shifted its focus to the basic research of F-T synthesis process technologies.4 6. Once this level of purity has been reached. Within the process boundary limits assumed here. After 1997. notably to research on slurry-phase F-T synthesis. the gas–solid fluid-bed F-T synthesis reactor technology was only developed when fluidization technologies became available in the chemical engineering field during the 1950s [17. 6. it is clear that the F-T synthesis process for processing syngas is rather straightforward.

Diesel from Syngas O2 CO2 Raw gas from gasification Purified syngas FTS loop Sulfur < 50 ppb Tail gas Sulfur recovery Sulfur product CO2 129 Water-gas Shift Acidic gas removal FTS crudes Diesel 70% Naphtha 20 % Product upgrading Dry gas Tail gas treatment Light hydrocarbons Oxygenates 4 % Water treatment FTS reaction water LPG 6 % Figure 6. but has been explored especially at Sasol to overcome shortcomings in the fixed-bed and gas–solid fluid-bed reactor technologies [18. especially at the design stage conceived during the early 1930s by Fischer. acidic gas removal. The FTS loop may be different due to technology choices. 18]. Figure 6. Syngas quality assumed here implies that modern gasification technology. The technical principles of these industrial reactors are discussed in the following text. product upgrading and water treatment steps are standard chemical processes. 8. These different reactor structures.1 Fixed Reactor Technology The Arge multitubular fixed reactor is the representative of the fixed-bed F-T synthesis reactor. A full integration regarding the efficient utilization of both mass and energy in the F-T synthesis-based process is important.and high-temperature F-T synthesis reactor technologies have been exhaustively reviewed [8. tailgas treatment. Today.2. form the core technologies in F-T synthesis.1 Simplified flow chart of a typical Fischer–Tropsch process. 6. 17. Although the fixed-bed reactor was the earliest commercial design used for F-T synthesis. and was selected and developed into a first set of commercial facility in South Africa by Sasol in 1955 [4. 17. All of these developments on Sasol’s low. 21]. combined with corresponding catalysts. 18].2 represents a typical fixed-bed F-T synthesis reactor and the catalysts used to fill the tube bundles of the reactor. its application in the field of CTL or GTL has been limited due to certain major . such as entrained-flow gasifier with coal-water slurry feed is used.4. WGS.

2 The multitubular fixed-bed F-T synthesis reactor (a) and the catalyst particles (b) used in the ICC’s demonstration facility. . the fixed-bed reactor is expected to be a promising technology for processing syngas from biomass in plants with relatively small production capacities. (iv) the low efficiency of the catalyst due to internal transfer limitations in catalyst particles that generally are 2–5 mm in size. Robustness in operation and its resistance to contaminants such as H2S.45 intrinsic drawbacks.2–0.130 Diesel from Biomass Figure 6. (iii) the complexity and demanding labor intensity of the procedures to replace the catalyst. This is partly the reason why Shell has selected the improved tubular fixed-bed reactor to conduct their SMDS process (12 000 bbl dÀ1) in Bintulu. In spite of these drawbacks. . where all the H2S is adsorbed by the top layers of catalyst. and (v) the limitations of operations to a single train capacity mode. including: (i) the nonuniform temperature profile along the fixed bed. (ii) the large pressure drop that occurs across the fixed bed. which makes it more compact and suitable for wax production. The particle size was 3 mm. and the estimated effective factor 0. The absence of a wax and catalyst separation problem. this is essentially equivalent to a guard bed for the remaining catalyst. Malaysia [23]. as the comparative advantages of smaller scales can then be harnessed over other large-scale reactor types: . due to difficulties in achieving an efficient reaction heat removal.

2.3 The fixed fluidized-bed F-T synthesis reactor operated above 320 C at about 25 bar.2 Gas–Solid Fluidized-Bed Technology The fluidized F-T synthesis reactor technology was initially developed in the US during the 1950s [16–18]. This technology implements a high-temperature operation mode with typically fused iron catalysts to produce only light hydrocarbons as the target products. insufficient information has been provided on these operational issues . The conceptual scheme of the latest version this type of the reactor is shown in Figure 6. In particular. 17].3. A fused iron catalyst is used. and then transferred to Sasol as the design of one of the reactor types selected for the Sasol-I plant [16. Compared to the fixed-bed version. gas–solid fluidization inside the reactor may be a severe problem due to equipment and catalyst damage. with the particle size range typically 50–300 mm. Issues of a hightemperature (>320 C) operation may cause significant concern in materials used for the manufacture of such huge vessels. The heavy products must be avoided in this type of the operation mode in order to ensure adequate gas–solid fluidization. To date. Figure 6.4. the major advantages of this type of reactor used in F-T synthesis include: (i) possible high throughput for a single reactor up to 20 000 bbl dÀ1.Diesel from Syngas 131 6.

including Sasol. and on-line catalyst replacement – evoked the interest of numerous players in the field. The catalyst used in this type of reactor has not progressed beyond the fused iron catalyst originally developed in the US during the 1950s and improved upon incrementally at Sasol. namely that any contaminants (e. fixed-bed. This one significant limitation to the productivity of the catalyst is normally monitored by determining the amount of target products produced per ton of catalyst consumed in the operation. The catalyst concentration in the slurry was used at a volumetric percentage of 25. The run lasted for 1241 h. static-bed and moving-bed processes in an effort to develop an F-T synthesis pilot plant [16]. allowing longer reactor runs. The arrangement of a typical slurry. H2S) can spread instantly over the catalyst inside the slurry. and the reaction temperature ranged from 242 to 276  C. the syngas H2/CO ratio was 1 : 1. leading to its .3 Slurry-Bed Reactor Technology Although the earliest slurry-bed studies can be traced back to studies of Fischer in 1932 [18. The obvious advantages of the slurry-phase process – namely a higher capacity. and (vii) the cost of a slurry reactor train being 25% less than that of a multitubular fixed-bed reactor.4. promoted by copper and alkali with weight ratios of 100 Fe/10 Cu/1 K. (iii) a fourfold lower catalyst consumption per ton of product as compared to other reactor types. a low temperature gradient inside reactor. China) to further develop the slurry-bed reactor processes.g.8 to 17 atm. 6. However. it was in the 1950s that the Bureau of Mine (US) in the US first investigated options such as fluidized-bed. In contrast. (v) a high selectivity of the slurry reactors towards the production of desired products. and operated at the low space velocity of 345 hÀ1. as significant catalyst loss is observed as a result of carbon deposition-related factors. An iron catalyst was used. ExxonMobil (Irving. (iii) enhanced heat transfer to maintain a better temperature control in the catalyst bed. obvious drawbacks exist in this type of gas–solid fluidized-bed operation mode for F-T synthesis. and (iii) high-temperature requirements leading to severe mechanical wear and tear of the vessel and all internal parts of the reactor. The unreacted catalyst was suspended in a synthetic diesel oil fraction that was characterized by a boiling point of approximately 300  C. Energy International (Florida. the reactor pressure varied from 6. 34]. Sasol undertook such optimization steps to enable the processes to be conducted under much more severe operational conditions and higher temperatures.. and Synfuels China (Taiyuan. In fact. USA).bed reactor is shown in Figure 6.2. leading to a low yield of desired products. the higher temperature used to conduct F-T synthesis operations remains an important problem to this date.4. the main disadvantage of this system is obvious. (ii) a pressure drop of the reactor which is about fourfold lower than that in fixed-bed F-T synthesis reactors. (ii) catalyst as well as mechanical damage due to severe physical conditions.132 Diesel from Biomass (ii) on-line catalyst replacement. (vi) mild operational conditions that ensure lower catalyst losses during fluid operations. Rentech (Colorado. USA). The oil circulation demonstration unit was of 3 m3 in capacity. and (iv) an absence of major intraparticle transfer limitations. The advantages of slurry over multitubular reactors include: (i) a more isothermal and higher operation temperature. (iv) the on-line removal or addition of catalyst. USA). Notably. including: (i) a high methane selectivity.

Another disadvantage that became evident during the scale-up phase is the vigorous movement and collision of catalyst particles that occur in slurry-type reactors. at least double productivity may be achieved due not only to . as such wear and tear produces micron-sized catalyst particles that in turn cause a significant increase in the viscosity of the slurry phase [9. Despite these problems. 35]. Typically. This ultimately leads to catalyst erosion and attrition.4 Conceptual scheme of (a) the slurry-bed F-T synthesis reactor. Today. the productivity of C3 þ hydrocarbon products can reach 400 tons per ton of iron-based catalyst consumed. recent progress in the high-temperature (260–290  C) slurry F-T synthesis developed at Synfuels China has pushed productivity to even higher levels. thereby improving the catalyst’s attrition performance parameters. The decrease in catalyst particle size may make separating the catalyst from the wax product extremely difficult.Diesel from Syngas 133 Figure 6. Notably. the slurry F-T synthesis process has been shown overall to have the highest productivity per ton of catalyst consumed. additional resources are required to separate the heavy wax from the slurry. Panel (b) shows the slurry F-T synthesis catalyst used at the Synfuels China’s pilot plant facility. Consequently. Particle sizes ranging from 40–200 mm are mainly used for the lowtemperature slurry F-T synthesis rapid deactivation. and this leads to dramatically increased downstream processing costs. as compared to any other type of reactor. for a conventional slurry-phase process operated in a low-temperature mode (ca. and (b) the associated catalyst particles. 17. 220–250  C).

and ICC/Synfuels China. 6. 18. Synfuels China Ltd. In addition to the conventional Arge fixed-bed and Synthol fluidized processes. The plant in Bintulu. naphtha. 22]. and (iv) an easy retrofit to both CTL and GTL processes.3.1 Sasol F-T Synthesis Process Since the commission of Sasol I in 1955. The development history of Sasol F-T synthesis processes is available in comprehensive reviews [5. Sasol has been actively developing F-T synthesis processes.3. the operating temperature of the HTSFTPÒ process is increased to approximately 275  C by using SynFT-I. The diesel fuel thus produced is proven to be a premium fuel characterized by a high cetane number (>75). a head-to-head comparison of HTSFTP-produced fuel with conventional diesel in engine tests showed that all emission parameters of the former . The wax is then hydrocracked or hydroisomerized to produce diesel fuels and other products. 17. reactor scale-up. (iii) the production of high-quality F-T synthesis syncrudes with very low oxygenates. In addition to its earlier fixed-bed reactor process (MFT). and wax. Notably. 9. a company evolved from ICC. The main characteristics and achievements of these processes are discussed in the following sections. uses natural gas as feedstock to produce diesel. an improved version of the Synthol process and later an original fixed fluidized-bed process (SAS) were developed in the period between 1980 and the 1990s. Shell.3 ICC (Synfuels China) HTSFTP Process The Institute of Coal Chemistry of the Chinese Academy of Science (ICC) has been conducting R&D in F-T synthesis technology since the 1980s. (ii) the efficient recovery of F-T synthesis reaction heat from the slurry-bed reactor as steam up to 30 bar. 6.2 Shell SMDS Process The Shell Middle Distillates (SMDS) process uses a cobalt catalyst with a high selectivity towards wax [23]. 6.3. the high-temperature slurry-bed F-T process (HTSFTPÒ ) originates from this research.4. which was commissioned in 1993.3 Major F-T Synthesis Process Development Endeavors The major developers of commercial projects in the CTL/GTL field include companies such as Sasol.4. Malaysia. 6. Moreover. and process integration at the pilot plant and demonstration scales [25–33].4. The major advantages of the HTSFTPÒ process over conventional low-temperature slurry-bed processes include: (i) a lower (about one-fourth) solid catalyst charge due to the ultra-active F-T synthesis catalyst being used. The catalyst used in this latter process is a proprietary iron high-temperature catalyst (named as SynFT-I) which was recently tailored for the high-temperature slurry-bed reactor [33]. Compared to the conventional low-temperature slurry-bed process. has carried out systematic proprietary catalyst development. Sasol also developed its proprietary slurry-bed reactor process during the 1990s.134 Diesel from Biomass the use of highly efficient F-T synthesis catalyst formulations but also to improved slurry reactor systems [33]. especially acids (about one-third). The composition and the preparation issues of the latest catalyst remain confidential.4.

g. waste.07) per cubic meter of coal-based syngas. Such systems are highly efficient and fuel flexible (e. In Europe. which showed highly competitive economics due to high oil prices. or biomass). hydrogen. The results have proved to be very positive for the new F-T synthesis technology developed at Synfuels China.45 RMB (US$ 0. A case-by-case study by Yamashita et al. such that the final diesel product was achieved. and (iv) the price of oil. or heat. One noticeable trend in the field of BTL has been the application of poly-generation or co-production strategies in order to leverage economies of scope and economies of scale [37]. [37] has indicated that integrated poly-generation processes might improve the economics of the whole ‘energyplexes’ with a syngas-based. In particular. The oil market in 2008 was a favorable factor to F-T synthesis projects. and separation of the catalyst from the wax proved to be highly efficient. The second phase is an expansion project designed to increase the capacity of this plant to 200 000 tons of BTL fuels per year by 2010. and that an at least 8% reduction in oil consumption can be achieved. multiproduct system.Diesel from Syngas 135 were clearly improved. However. The slurry reactor operation demonstrated a very uniform distribution of temperature in the slurry bed. chemicals. Three F-T synthesis plants with capacities of 3500–4000 barrels per day were in the final stages of construction in 2008. In particular. (iii) the operational costs. natural gas. 6. biomassbased syngas is expected to be significantly more expensive than coal-based syngas because . most BTL plants are. Synfuels China has formed a strategic alliance with several energy companies.4 BTL Project Status Today. and coal. (ii) the raw materials cost.35–0. since which times one of these has undergone a first test run (operation for 23 days to detect process problems). with a very low methane selectivity of less than 3 wt%.0–1.4 g gÀ1 catalyst hÀ1.4. The economics analysis presented here is based on varying cost assumptions for cleaned syngas. most of the investigations into BTL are conducted within the framework of the European project RENEW. at the demonstration stage. entrained-flow and fluidized-bed gasifiers have been examined in this project. Both.5 Economics The economics of an F-T synthesis plant largely depends on: (i) the capital cost of construction. advanced gasification technologies and F-T synthesis processes have been implemented to generate various end products such as electricity. at most. which has been used to test various gasification processes and process configurations. the syngas cost assumption is based on 2008 price projections of 0. liquid fuels (diesel. etc. For example. with the aim of demonstrating the benefits of this technology. Another BTL project in Europe is the ECN/Shell project. 6. The iron catalyst reached a record hydrocarbon productivity of 1. coal.). multifuel. The first phase of the project was a 15 000 ton BTL fuels per year plant constructed in 2003. the Choren GmbH project is a 1 MW demonstration BTL plant for processing clean wood.05–0. a detailed techno-economic analysis and a large-scale plant simulation work have also been carried out [36]. In addition to the laboratory-scale experimental studies. which is supported by the European Commission [36].

it is safe to say that all unit operations in a biomass-to-diesel plant are commercially proven. the practical partitioning of the cost of syngas derived from coal can form the basis of an estimate of biomass-derived syngas.36 22 26. and about US$ 730 tonÀ1 (US$ 90. The data in Table 6.6 per barrel) with CTL. The syngas consumption is 5560 m3 tÀ1 of oil products when a technology such as HTSFTP F-T synthesis is used.6 Perspective Today. while the economics of BTL is also promising when the oil price is above US$ 100 per barrel. commercial-scale plants have been commissioned to .3 The cost of syngas in US$ per m3 syngas (CO þ H2) in US$ a Items Capital Feed to gasification Oxygen þ Operation Sum a Syngas cost from coal 0.03 0.103 m3.4 The cost of F-T synthesis final products (in US$) Items Capital Syngas FTS catalyst/others Operation Sum Syngas cost from coal 33. In a realistic cost estimate of biomass-derived syngas. It can be concluded that CTL. and that of biomass-based syngas about US$ 0.023 0.688 393.432 of the low energy value of the biomass feed for a gasification unit. Detailed studies taking all of these factors into consideration are still to this date not possible.68 44 40. With these quantities as a starting point.015 0.36 311. however. 6. and diesel) can be estimated relatively accurately. under crude oil prices above US$ 60 per barrel is economically competitive.056 mÀ3.136 Diesel from Biomass Table 6.6 per barrel) with BTL. For both CTL and GTL.4 show that the overall cost of F-T synthesis-derived products is approximately US$ 400 tonÀ1 (US$ 49.032 723.046 0. naphtha.72 572. both the F-T process and product upgrading costs (to LPG.408 Syngas cost from biomass 66.027 0.3 show that the cost of coal-based syngas is about US$ 0. The data in Table 6. one must consider the higher capital and operational costs incurred by the practitioner during the gasification process.018 0. dry biomass costs US$ 80 tonÀ1. Table 6. pending further improvement in biomass gasification efficiency.103 Coal costs US$ 40 tonÀ1.056 Syngas cost from biomass 0. and the overall high collection cost of biomass.

During the scale-up. as the key technology in converting syngas to diesel fuels. and environmental policies. will push catalyst design and development forward. Indeed. 20590361). the overall techno-economic performance of a BTL plant will derive from the many factors discussed in this book. yet the overall process integration and optimization will be conducted and more stringent requirements on catalyst development raised that. It might be reasonable to ask the question for the future development of syngas production: What is the optimal feedstock solution for the gasifiers typically operating above 1200  C? Fischer–Tropsch synthesis technology. With regards to technological aspects. active. Clearly. Future efforts in this direction should therefore be focused on identifying a more efficient way of producing syngas from biomass. It is of interest to note that almost all successful F-T catalysts are in fact nanostructures at the surface level. This implies that the findings of nanotechnology research will play an increasingly important role in future F-T catalyst development. In addition to the major direction for future F-T technology development – namely. Nanotechnology may lead to very different catalyst designs [12] on the basis of a fundamental understanding of F-T catalysis and process integration [33]. it should be pointed out that F-T synthesis technology does not differ greatly from that used for CTL and GTL. heavy oil) is the key issue to having a high efficiency in syngas production by using current high-temperature gasification processes. natural gas. and selective catalysts and more efficient reactor systems – the efficient recovery of heat released from F-T reactions clearly requires higher operational temperatures for F-T reactions.Diesel from Syngas 137 convert either coal. and syngas production does indeed leave space for further development. numerous unexpected technical hurdles will surely loom up. no integrated process has ever been developed commercially in the BTL context. whether the catalysts are created using a conventional or a nanotechnological approach. Acknowledgments The authors are very grateful to the following organizations: Ministry of Science and Technology of China (MOST) and Chinese Academy of Sciences (CAS). and this poses the major technical factor limiting BTL via F-T synthesis. the commercialization of BTL technology via biomass-derived syngas will be lagging behind the CTL or GTL schemes. However.or natural gas-derived syngas to diesel fuels. consequently. The future development of any large-scale BTL plant depends on government policy incentives. At this stage. for supporting the industrialization efforts of the F-T technology and the National Natural Science Foundation of China for fundamental research directions (20625620. this has not yet attracted much attention. as well as chemicals. due mainly to economic reasons. however. more stable. will be significantly optimized along with the CTL/GTL commercialization activities. this may require comprehensive considerations to upgrade the biomass feed stock to a high-energy density mode in a cost-effective manner. in turn. naphtha and LPG. crude oil prices. especially for biomass. for which the gasification concept mimicked from coal gasification does not represent a cheap cake for biomass processing. . it is fact that the low heat value of biomass compared to fossil fuel (coal.

and J. 1925. Schlesinger. Amsterdam: Elsevier. vol. 2002.B. Kukulj. 20. M. T. 158–224. pp. 2008. 11. Introduction to Fischer–Tropsch synthesis. 152. 2104. Gautam. M. vol. 2007. 2004. P. 1951. N. 163. 4. Comparing Fischer–Tropsch synthesis on iron. Davis.E. Storch. C. Industrial & Engineering Chemistry. J. Presented at Fischer–Tropsch Synthesis on the Eve of the XXI century (CATSA). R. Elsevier. J.138 Diesel from Biomass References 1. L. FT catalysts. Atkinson. Rud. Bartholomew. 17. vol. R.B.. Synthesis Gas as an Industrial Feedstock.L.E. International Petroleum Economics. 3.R. Occelli (eds). P. 17. 23. 1. Dry (eds). presented at SAE Technical Paper Series. 64. 121. 1984. M. D. 12. 177–200. Global Prospects of Synthetic Diesel Fuel Produced from Hydrocarbon Resources in Hydrocarbon Resources in Oil & Gas Exporting Countries. 533–600. 2007. Vertin. Xiao. B. 1–63.H. in The Fischer–Tropsch Synthesis. Crowell. Eberhardt. Int.E. Chem. 22.E. 6.zb. Schlesinger and H. Dry. Development of GTL and Related Technical Economic Analysis. Dry. Present and future applications of the Fischer Tropsch process. Dry. No. Anderson. Presented at 1st International Biorefinery Workshop. Presented at Preprints – Division of Petroleum Chemistry. 26. Stranges. W. 10. Occelli (eds). 1991. Storch. 7. 19. 47. 5. Kurevija. 2005. .-geol. Washington. Catalytic Aspect of Industrial Fischer–Tropsch Synthesis.H. Xie. English edition translated by Lessing. Dry.B. M. van Ree. Schulz. 4: Symposia.H. in Studies in Surface Science and Catalysis. M. USA. Anderson. Wang. On-Road Use of Fischer–Tropsch Diesel Blends. 23. 1981. vol. Zwart. Kruger Park. 746–749. M. A history of the Fischer–Tropsch synthesis in Germany 1926–45.P. Applied Catalysis A: General. New York: John Wiley & Sons. and H. 1474.E. Fischer. The Fischer–Tropsch and Related Synthesis.X. F. 47. Norton.and cobalt catalysts: the dynamics structure and Function. Dry. 276.Cai. 1981. and H. Davis and M. American Chemical Society.R. Goguen. M. Catalysis Letters. Upgrading Fischer–Tropsch Products. Leva.X. 1–3. Boudart (eds). 2004. 133–144.H. in Studies in Surface Science and Catalysis. 303–316. M. Amsterdam: Elsevier. Journal of Molecular Catalysis. C. Kou. M.W. Steynberg. 159–255. 13. N. Yang. K. Bartholomew. 163. The conversion of coal into oils. A. New York. Yao. Washington. in Studies in Surface Science and Catalysis. A. Techno-economic and environmental analysis of a thermo-chemical Biorefinery process for large scale Biosyngasderived FT-diesel production.-naft. Davis and M. vol. pp. Amsterdam: Elsvier. The Fischer–Tropsch Synthesis. 21. 2004. H. Guczi (ed. in Studies in Surface Science and Catalysis. 19. Ed. Rajkovic. p. pp. 2004. Fischer–Trospsch in Slurry Phase. Vol. 14. Lyons. and R. Golumbic. in Studies in Surface Science and Catalysis. 1951. van der Drift.E. 2005. 2. T. Clark. Z. pp. Steynburg and M. London: Academic Press. The Fischer–Tropsch Synthesis. pp. Fischer–Tropsch reactors. vol. Y. 16. Recent Developments in Fischer–Tropsch Catalysis. 152.E. vol. Angew. R.L. R. pp. A. Industrial and Engineering Chemistry. 1990. N. Boerrigter. 2007. B. B. C. 9. pp. 15. Springer-Verlag. S. 8. 1982.E.D. in Studies in Surface Science and Catalysis. Hydrocarbon Processing. Breman.). 64–195. and N. London: Ernest Benn Limited. Amsterdam: Elsevier. Anderson and M. C. A. 13. The Fischer–Tropsch process: 1950–2000. M. H.H. Amsterdam. NY. DC. 1–27. Aqueous-Phase Fischer–Tropsch Synthesis with a Ruthenium Nanocluster Catalyst. Benson.P. M.H. 1982. A. 1955. 1999. Dry.E.D.H. M. G. 18.N. 1 November 2000. and B. Sasol’s Fischer–Tropsch experience. 7. Dry. Recent technological developments in Fischer–Tropsch catalysts. Dry. 152. Technology of the Fischer–Tropsch Process. Steynberg. in New Trends in CO Activation. 79. and D.H.

2005. Li.G.-Y. Yang. Zhao. Zhao. Hao. 30.K. Xu. Activation of Precipitated Iron Fischer–Tropsch Synthesis Catalysts (part a). Y. 26. H. 2003. Li.-Y. Li.-W. 1995. Xu. Xiang. Presented at Natural Gas Conversion V. 2005.-W. 35. Zhong. 156. 81. 33. H.-W. Wang. 1998. Heterogeneous modeling for fixed-bed Fischer–Tropsch synthesis: Reactor model and its applications. Yang. Slurry phase Fischer–Tropsch synthesis over manganese-promoted iron ultrafine particle catalyst. Zhong. Datye. 29. Joint Research Center. H.-N. Catalysis Reviews: Scientific Engineering. J. Y. Bai. 36. Current status and industrial applying prospect of coal indirect liquefaction technology. Y. European Commission. X. 58. Y. 2003.-Y. Y.J. K.-J. J. Y. S. 2006. 25. Industrial and Engineering Chemistry Research. Y. Yang. The Netherlands EUR 21745 EN. Jager. Y. Xu. Coulter. Chemical Engineering and Technology. Wang. 5066–5090. Xu. J. 30. Zhang. Han. 34. K. 42. H. 185–207.-W. 867–875.D. B. Y. Lu. 1980.-N. Shroff. Xiang. Y. Zhang. Sault.W.-J. Shanxi Science and Technology Press. Kavalov and S. W.Z. Zhang. Journal of Catalysis. 17.-Y. Ma.Y. Effect of reaction conditions on the product distribution during Fischer–Tropsch synthesis over an industrial Fe-Mn catalyst. Xiang. Y. Coal-based Synthetic Liquid Fuels. Peteves.-L. 195–213. Y. 2453. Ralek. B. Modeling of catalyst pellets for Fischer–Tropsch synthesis. Yamashita and L. Status and Perspectives of Biomass-to-Liquid Fuels in the European Union. 30. 2007. IEC Res. and B. Barreto. M. 4324–4335.-W.X.-Y. 31. J. and Y. Koler. Journal of Chemical Industry & Engineering. Fuel. 214. Petten. Y. A. Li. G. Harrington. 27. Xiang. 37. Kalakkad. electricity and liquid fuels.E. 225–274. Xu.-W. Li.. Y. Wang. Coal to Liquid (CTL): Commercialization Prospects in China. Detailed kinetics of Fischer–Tropsch synthesis on an industrial Fe-Mn ultrafine particle iron catalyst. . 28. 2002. Kinetics modelling of Fischer–Tropsch synthesis over an industrial Fe-Cu-K catalyst. Applied Catalysis A: General. Energy. Jackson. Y. Zhang. 77–86. and B. Y.-L. Fuel. H. and B. Chemical Engineering Science. Y. dong. 2001. 21.-J. The Fischer–Tropsch Synthesis in the Liquid Phase. 56. M. N.D. and A. Y. and B. Y.-L.S. D. Ji. Energyplexes for the 21st century: Coal gasification for co-producing hydrogen. 32. 82. Zhang.-W. Studies in Surface Science and Catalysis.-P.W.-N. 40. and B.D. 2001. Developments in Fischer–Tropsch technology. Li. 1577–1581. Yang.W. Kobel and M.Diesel from Syngas 139 24. 1993. 2003.-J. B.Q. L.B.S. 1157.


As a result. but now production in the US is growing rapidly. logging. an important advantage of the diesel engine was that it could use the portion of crude petroleum that was not needed for gasoline and included byproduct streams of gasoline refining. 2007). there is a need for alternatives so that the key industries in the world Biomass to Biofuels: Strategies for Global Industries and Hideaki Yukawa Ó 2010 John Wiley & Sons. Hans Blaschek e . and reliability are important. As a result. diesel fuel is generally produced from higher-boiling point fractions of crude petroleum than are used for gasoline. They are used almost exclusively in the agricultural. Europeans recognized the potential of producing diesel fuel from biomass several years before the US. durability. Nasib Qureshi. Diesel engines require a fuel that easily auto-ignites under the high temperature and pressure conditions found in the diesel cylinder. In the United States. However. and it incorporates the long. 1988). construction. diesel fuel is frequently more expensive than gasoline and must meet equally stringent quality requirements. nowadays. but in Europe more than half of all new cars sales are diesel-powered (Valdes-Dapena. As the cost of petroleum increases and easily-extracted petroleum becomes more difficult to find.1 Introduction Diesel engines are the prime mover of choice for applications where fuel economy. Ltd Edited by Alain Verts. When it was originally introduced. diesels are rarely used in passenger cars. straight-chain alkanes that provide good ignition characteristics in the engine (Heywood. Fuel volatility is less important than for gasoline-fueled engines. diesel fuel costs were low and diesel engines inexpensive to operate. because in diesel engines the fuel is not required to vaporize and mix with the air at ambient conditions outside the engine cylinder. trucking and railroad industries.7 Biodiesel from Vegetable Oils Jon Van Gerpen 7.

Moreover. Moreover. Barsic and Humke (1981) as well as Humke and Barsic (1981) tested crude. the performance and emissions returned to their original values. Deposits on the injection nozzles were identified as the source of this problem and when the deposits were removed. Suda (1984) reported that in Brazil over 10 000 h of operation had been accumulated on five vehicles powered with Caterpillar engines. nitrogen oxides in the exhaust gas show a tendency to decline when vegetable oils are substituted for diesel fuel. while the higher fuel density increased the fuel energy delivery so the energy was equal or greater than that of conventional diesel fuel. Nonetheless. carbon monoxide increases slightly at light and medium loads and decreases at high loads. the fact that fat oils from vegetable sources can be used may seem insignificant today. when using either these pure vegetable oils or their blends with diesel fuel. It also includes a description of current biodiesel production technology. and that integrates easily into the existing diesel fuel infrastructure. at least for short periods of time. they provide the additional benefit of decreased net carbon dioxide emissions.142 Diesel from Biomass economy have the fuel needed to continue their operations. Humke and Barsic (1981) noted that engine performance decreased and emissions increased as the test progressed. This chapter describes the ways in which vegetable oils have been used in diesel engines. and explains why biodiesel has emerged as the most satisfactory way to use this renewable resource. Diesel expressed this view by the following notable statement “. in diesel engines. at equal energy inputs. Interestingly.” (Diesel. Although the first use of vegetable oil in a diesel engine is frequently attributed to Rudolf Diesel himself. due to reduced injection pump leakage. the test engines produced equivalent power in spite of the lower energy content of the fuel. 1984). 7. 1912) Investigation of the direct use of vegetable oils as a substitute for diesel fuel became an urgent task when oil supplies were short and prices rose during the late 1970s and early 1980s. the total amounts of unburned hydrocarbons in the exhaust gas change relatively little at light and medium loads. . but excessive piston ring and liner wear were observed at high loads with vegetable oil (Suda. Investigations on prechamber-type diesel engines demonstrated that a similar performance could be achieved with conventional diesel or crude-degummed vegetable oils. Nonetheless. Vegetable oils provide a fuel source that utilizes well-known technologies.2 Use of Vegetable Oils as Diesel Fuels Vegetable oils have properties that make them suitable for use. since the plants that provide the oil can recycle atmospheric carbon dioxide. The greater viscosity of the vegetable oil fuel increased fuel mass delivery. Another interesting observation is that particulate emissions increase slightly at high loads but are variable at other loads. since fuels derived from agriculture could provide energy after natural sources are exhausted. Diesel recognized early on that vegetable sources of oil could become as important as mineral oils. Knothe (2005a) has shown that Diesel merely observed the use of peanut oil in one of his company’s engines at the Paris Exposition in 1900. despite a measurable increase relative to diesel at high loads. but such oils may perhaps become in course of time of the same importance as some natural mineral oils and the tar products are now. without major . What is more. . undegummed sunflower and peanut oils in a direct injection engine. They found that. Similarly. For example.

Between 20 % and 30 % butanol was added to promote the formation of a stable microemulsion. No. However. although this wear appeared to be lower than what had been previously observed with high-sulfur Brazilian diesel fuel. the test was too short to provide any information on engine durability. In addition.77 mm2 sÀ1) increases the amount of fuel that can be injected. offer the additional benefit that they can lower the temperature during combustion and consequently of reduced emissions of nitrogen oxides (NOx) (Goering et al. No.5 on the Bosch scale. upon disassembly of the engines. Fishinger et al. deposits in the piston ring grooves caused ring sticking and engine failure. an increased propensity to deposit build-up on the nozzle tips and on the piston and liner were also noted by Baranescu and Lusco (1982).0 to 8. The only concern noted resulted from an inadvertent substitution of No. with the exception of a plugged injection nozzle which was thought to have been caused by excessive carbon build-up remaining from the first 200 h of the test. Since ethanol and butanol have low cetane numbers.. when vegetable oil was used as a fuel. these effects were usually attributed to the high viscosity of the vegetable oils. Likewise. when degummed and dewaxed sunflower oil is used. 2 diesel fuel. In a similar experiment. Emulsions with liquids such as alcohols and water. 2 diesel fuel to gel at low temperatures. They found that. (1982) evaluated emulsions of ethanol and soybean oil as substitutes of petroleum diesel fuel. the piston ring and liner wear were found to be higher than would have been expected if conventional US diesel fuel had been used. The higher viscosity of the fuel (6. Crookes et al. Goering et al. 2 diesel fuel. this fuel provided approximately 6 % better thermal efficiency than No. 1 diesel fuel is composed of hydrocarbons with lower boiling points. . This effect. and is most commonly used in winter to protect against fuel gelling. and the resultant viscosity increase. Ziemke et al. which have high enthalpies of vaporization. Nevertheless. this was attributed to an excessive internal carbon build-up and to a large increase in crankcase oil viscosity. combined with the higher thermal efficiency. (1983) operated a direct injection diesel engine for over 1000 h on blends of soybean oil and diesel fuel. 1 diesel fuel in the blend.Biodiesel from Vegetable Oils 143 problems. with only 82 % of the energy content of the diesel fuel. (1992) collected performance and emissions data from a diesel engine operated with emulsions of diesel fuel and vegetable oil with 10 % water. 1 diesel fuel could be used satisfactorily in a Detroit Diesel 6V-71 two-stroke diesel bus engine. caused the smoke level of the bus to increase from 2. Notably. 2 diesel fuel for No. as it does not contain the waxes that cause No. It is particularly worth noting that elevated deposit levels were frequently observed in these studies. This change. The first 200 h of the test were conducted on a blend of 50 % soybean oil and 50 % diesel fuel. During this portion of the testing. (1981) reported that a 20 % blend of waste vegetable oil in No. This was probably due to overfueling caused by the higher density and viscosity of the heavier fuel. a large amount (10 %) of alkyl nitrate to serve as cetane improver was added to raise the cetane number to 40. The last 800 h of the test were conducted with 33 % soybean oil and 67 % diesel fuel. emulsions of vegetable oil with low-viscosity liquids were investigated as an alternative to reduce the overall viscosity of the fuel. As a result. This portion of the test was completed satisfactorily. results in engine power that is similar to that achieved with No. 2 diesel fuel is the usual grade of fuel used by most over-the-road trucks. Pestes and Stanislao (1984) observed that. 1982). the engine power and efficiency declined.

144 Diesel from Biomass in this particular system. Interest in the US was chiefly evoked by the formation of the National Soydiesel Development Board in 1992. 1937) for the use of ethyl esters of palm oil as diesel fuel. It is these attributes that have provided a large part of the incentive for substituting biodiesel blends for diesel fuel in urban buses (Schumacher. Free water or alcohol would cause corrosion and performance problems in the engine. (1995) tested an emulsion of palm oil methyl esters. including fewer injector deposits and less wear than with ordinary diesel fuel. The problems of excessive deposits and in-cylinder wear that result from the use of vegetable oils in engines caused most researchers to seek ways of utilizing vegetable oils other than as triglycerides. After that time. such fuel formulations have never achieved widespread use. In spite of the apparent success with the use of emulsions. where XX represents the percentage (usually by volume) of biodiesel. are shown in Table 7. These authors concluded that there was no doubt that an ester prepared from sunflower oil could constitute an appropriate substitute fuel for direct injection diesel engines. and compared with the emissions measured when using diesel fuel (Sharp et al. By chemically converting the triglycerides in the oil to alkyl esters (which can be readily achieved by reacting the oil with a simple alcohol). due mostly to concerns about separation of the water or alcohol. The . there appears to have been little interest in alkyl esters until the early 1980s. It should be emphasized that the smoke level and NOx emissions were lower for the vegetable oil than for diesel fuel.1.. In particular. particularly at low temperatures. and particulate emissions. the delays observed with vegetable oils were the shortest. 1982). followed by that attained when using conventional diesel fuel. 2007). which changed its name shortly afterwards to the National Biodiesel Board. Testing has demonstrated that biodiesel and its blends with diesel fuel provide very significant reductions in exhaust CO. Pischinger and Falcon (1982) confirmed this result by noting that soybean oil methyl esters showed a high degree of compatibility with existing diesel engines. 2 diesel fuel. Similarly. The emissions for three engines operated on B20 and B100. unburned hydrocarbons. 2000). These observations were all consistent with the cooling effect of water vaporization that ultimately results in a slowing down of the precombustion reactions that control the length of the ignition delay. 1995). Chavanne at the University of Brussels was granted a patent (Chavanne. diesel fuel and water and found satisfactory operation. Knothe (2005a) has identified early research performed in Belgium and the Belgian Congo as the first demonstration that alkyl esters could be used in diesel engines. when researchers in South Africa reported on transesterified sunflower oil (Hawkins and Fuls. it was possible to obtain a fuel with properties that are much closer to those of No. Sii et al. G. Biodiesel blends are commonly denoted by BXX. while those ignition delays observed with the emulsions were the longest. 2005a). Notably. with the remainder being No. the ignition delays of the emulsions were similar to those of the original fuels. and that they were even lower for the emulsions. although some minor differences persisted. 2 diesel fuel. Alkyl ester production plants were built in Europe during the late 1980s. but occasional use has been noted as early as 1984 (Van Gerpen et al. This work was carried out with the intent of making the African colonies self-sufficient in fuel. The first use of the term ‘biodiesel’ is unclear. These alkyl esters have become known as biodiesel (Knothe..

7 À36.. such as the isentropic bulk modulus and the speed of sound.9 À20. The reasons for this increase are still not yet fully understood.80 À49. when the PM is separated into its two main fractions – the volatile organic fraction (VOF) and the soot fraction – it becomes apparent that the VOF actually increases for biodiesel. can cause the fuel injection timing to advance which can cause the NOx to increase (Tat et al. 2006). Biodiesel was observed to lower in-cylinder soot concentrations which are responsible for the radiation heat transfer. On the other hand. The PM emissions are substantially reduced with biodiesel.4 À83.4 þ 10.0 À21. Krahl et al.3 0 À74.30 Soot À60.8 þ 31. Another reason that has been proposed is the elevation of combustion temperatures that results from reduced radiation heat transfer when biodiesel is used (Cheng et al. However.1 Exhaust emissions for B20 and B100 (Sharp et al. and the resultant higher gas temperatures would be expected to raise NOx levels through the Zeldovich chemical mechanism (Heywood. 2007). The large decrease in soot. 2008).8 À62. PM ¼ particulate matter. While the regulated emissions varied.8 HC ¼ unburned hydrocarbons.7 À38. this is compensated by the soot fraction. In turn. (2007) compared the emissions resulting from the use of rapeseed oil with those attained when using methyl ester of rapeseed oil.Biodiesel from Vegetable Oils Table 7.3 À45.3 À14. the low volatility of biodiesel prevents a fraction of the fuel from vaporizing.5 þ 13.90 À28.6 À17. Apparently. they generally confirmed the results in Table 7. VOF ¼ volatile organic fraction engines were tested using the transient test procedure used by the Environmental Protection Agency to certify truck engines (CFR. which decreases to levels that are even greater than those of the overall PM. CO ¼ carbon monoxide. as demonstrated in this experiment where one particular engine exhibited a 49 % decrease.6 À8. 1988). could be an important advantage of biodiesel. and conventional diesel fuel. Nonetheless. and particulate matter (PM) levels. In a recent study. this unburned fuel is swept out into the exhaust where it can be captured as PM. Tat et al. CO. changes in the physical properties of the fuel.9 B100 Cummins B5.2 À32.3 À3. one surprising result was the 30-fold higher mutagenicity (or cancer-causing potential of the exhaust) of rapeseed oil as compared to biodiesel and diesel fuel.9 À20.1 % for B100. The data show that all three engines have major reductions in exhaust unburned hydrocarbon (HC)...3 þ 3. 2000.30 þ 1.0 À13.3 À71. 2000) Test engine Test fuel HC CO Transient emissions NOx g hp À h 145 Particulate emissions Total PM VOF þ 42..0 Cummins N-14 B100 Cummins N-14 B20 Detroit Diesel Series 50 B100 Detroit Diesel 50 B20 Cummins B5.2 þ 11.9 B20 a À95.1 þ 4. NOx ¼ oxides of nitrogen. nitrogen oxide levels rise by 4.8 þ 14. .4 À23.60 þ 4.3 þ 13.3 % to 13. Nevertheless. as the soot is much more difficult to oxidize in a particulate trap.1 for biodiesel and diesel fuel. even if accompanied by an increase in VOF.4 À38.3 À7. this results in fuel deposits on the combustion chamber walls.7 À14.

although this generally refers to alkane-rich fuels produced from carbohydrates. the European Specification EN14214. Normal alkanes are present in conventional diesel fuels. Huber et al. The product.. is renewable diesel this is generally used to designate fuels other than alkyl esters that are produced from vegetable oils or animal fats. 7. thus producing a fuel that is rich in normal alkanes (Furimsky. 2000). the energy content of renewable diesel will typically be slightly lower than that of a conventional diesel fuel. Green diesel is another term that is commonly used for designating biomass-based diesel fuels. 2008. there is considerable variation regarding the definition of the term. The fuel may be either coprocessed with a conventional petroleum stream (Conoco/ Philips. Transesterification is a type of chemical reaction that. wood oils. consists mostly of n-alkanes and has a cetane number of approximately 100. Because a great part of the oxygen that was originally present in the oil or fat has been removed. CEN. 2005). Because there is currently no specification for these fuels. following processes described by Dumesic and Huber (Huber et al.3 Renewable Diesel The term biodiesel is widely used around the world to denote alkyl esters produced by the transesterification of plant oils and animal fats with simple alcohols. As a result..4 Properties Vegetable oils and animal fats are composed of triglycerides. in this instance. One class of renewable diesel fuels is constituted by fuels that are produced at a petroleum refinery from vegetable oils and animal fats. The CANMET Energy Technology Centre in Ottawa. or may be processed and maintained as an identifiable stream (Rantanen et al. Canada. 2007. when renewable diesel is blended with conventional diesel fuel it is difficult to distinguish. animal fats. These fuels are alkane-based.. 2007). like renewable diesel.. which can perhaps be best defined as a glycerin backbone to which three fatty acid chains are attached. West et al. has developed technologies for hydrotreating vegetable oils. 2006. 1998). Nevertheless. alkanes tend to have low densities. The difference is small enough that any mileage difference would remain essentially unnoticeable by consumers. One additional drawback of renewable diesel is that n-alkanes tend to be relatively poor lubricants and to have poor cold-flow properties. the energy content of the renewable diesel fuel is significantly higher than that of the original vegetable oil or biodiesel. 2008). However. but which is not as well defined. but the feedstock employed to manufacture them is a simple sugar rather than an oil or a fat. The refinery removes oxygen and adds hydrogen. 2008.. 2008). this can usually be attenuated by the use of additives. NaOH. and many other international specifications (ASTM.146 Diesel from Biomass 7. hence. known as SuperCetane. and tall oil (a byproduct of paper production) to produce a high-cetane diesel fuel additive (Monnier et al. This is the definition that is used in the American Society for Testing and Materials (ASTM) specification D 6751. involves reacting a triglyceride molecule with an excess of alcohol in the presence of a catalyst (typically: KOH. . Carlson et al. Another term that is frequently used.. 2005. The biomass-derived alkanes provide good cetane numbers and are compatible with existing engines and fuel distribution infrastructures.

and polyunsaturated fat contents. 2005).e. An exception to this is palm oil. The transesterification reaction of a triglyceride with methanol to produce methyl esters and glycerol is shown in Scheme 7.2. A fatty acid with one double bond is monounsaturated. KOH.Biodiesel from Vegetable Oils 147 Scheme 7. the number of carbon–carbon double bonds) is the parameter that has the most significant impact on the cetane number. but ethanol and other higher alcohols can also be used. it is the methyl esters that become biodiesel.. The amount of saturated alkyl esters (i. 2007. The alcohol shown is methanol. Animal fats tend to have higher saturated fat contents than most vegetable oils.. Most of the properties of a biodiesel are determined by the relative amounts of the four or five alkyl esters that are present in that particular fuel. Fatty acids with no double bonds are said to be saturated. a 16-carbon chain with no double bonds. and are not susceptible to the oxidative attack of the double bonds that occurs in unsaturated fatty acid chains. where XX designates the number of carbon atoms in the fatty acid chain and Y the number of double bonds. Studies are currently under way to develop solid catalysts (Xie et al. a saturated fatty acid. This reaction is used to produce biodiesel from oils and fats. with esters of higher alcohols being slightly less.1. For example. whereas a fatty acid with two or more double bonds is polyunsaturated. which means that in practice they must be kept above their cloud point temperature. 2005. . 2006) that could be used with packed beds so that the fuel emerging from the bed is not contaminated with catalyst residue NaOCH3. which are dissolved in the alcohol before mixing with the oil. the saturated alkyl esters tend to solidify. The most common catalysts are NaOH. and can also cause crosslinking and polymerization that generates harmful sediments and varnish deposits. 2003.1 The transesterification of triglycerides. and NaOCH3. Here. The key differences between the fatty acids are the lengths of the carbon chain and the number of carbon–carbon double bonds. which contains a high amount of palmitic acid. 2005b). The fatty acid chains are designated in Scheme 7. Mbaraka and Shanks. Mbaraka et al. Most common oils and fats contain relatively few different fatty acids. Saturated alkyl esters have high cetane numbers (Knothe. This oxidation reaction can result in the breaking of the fatty acid chains into shorter chain acids. Fatty acids are commonly denoted by XX:Y. oxidative stability and cold-flow properties of the final fuel.. 16:0 designates palmitic acid. The fatty acid profiles of some commonly used oils and fats are listed in Table 7. and R3. At low temperature.1 as R1. which typically have higher mono. R2.) to produce glycerol and alkyl esters (Van Gerpen. Methyl esters have the highest cloud point temperature. These amounts are determined by the fatty acid profile of the oil used as the primary raw material. etc.

7 12.5–2 16.7 — — 0.4 12.15 0.5 — 15–17 — — — 1.4 20–30 19–49 50–65 73–84 23–35 16.4 1.3 17. (1992) Salunkhe et al.2 11.1 10–13 12–18 20–25 3.2 1.3 3.1 14.5 27.7 24. and 45 % C12:0 . (1992) University of Idaho (2008) Adebowale and Adedire (2006) Linstromberg (1970) Tat and Van Gerpen (2003) Diesel from Biomass Oil or fat Soybean Corn Peanut Olive Cottonseed Sunflower High-linoleic safflower High-oleic safflower Canola High-erucic rapeseed Oriental mustard (Pacific Gold) Yellow mustard (Ida Gold) Butter Lard Tallow Palm Palm olein Palm kernela Camelina Jatropha Linseed oil Yellow grease a Palm kernel oil also contains 3 % C8:0.3 35–40 8.1 1–10 4–8 1.8 9.6 5.2 — 25–60 0. (1992) University of Idaho (2008) University of Idaho (2008) University of Idaho (2008) University of Idaho (2008) Linstromberg (1970) Linstromberg (1970) Linstromberg (1970) Sapuam et al.6–4.8 47.3 13–19 15.8 — — — — — — 3.5 0–1 — — — — 35.0 9.0 2–4 12.148 Table 7.8 25–40 54.2 Fatty acid composition of various oils and fats 14:0 — 1–2 — — 0–2 — — — — — — — 7–10 1–2 3–6 1.5 16:0 18:0 18:1 18:2 18:3 20:1 22:1 Reference Linstromberg (1970) Linstromberg (1970) Linstromberg (1970) Linstromberg (1970) Linstromberg (1970) Salunkhe et al.8 2.2 9.8 28–31 40–50 37–43 39–45 40.5 10.0 5–11 trace — trace trace — 1 — 13.2 2–10 4–8 4.0 2.0 12.2–0.3 0.1 20.1 17.8 0.6 — — — — — — 14.9 13.9 32.1–2.4 2–5 2–5 2–3 2–3 1–2 4.3 1–3 2.8 — — — — — — — — — — — 50.5 55–81 11–19 21. 3 % C10:0.45 1–2.8 24–26 28–30 24–32 40–46 41.3 6–10 8–12 8–9 9–10 20–25 7.7 — — — — — — — — — 7. (1996) University of Idaho (2008) Salunkhe et al.2 7–42 74–79 59.8 1. (1992) Salunkhe et al.7 50–60 34–62 20–30 10–12 40–50 72.1 6–9 5.3 4–7 17.5 7–13 2–3 7–11 10.

500 ppm for S500 grade 360 max Units  C % volume  C % volume mm2 sÀ1 % mass  C % mass mg KOH gÀ1 % mass % mass % mass  C ppm (mg gÀ1) ppm (mg gÀ1) hours ppm (mg gÀ1) seconds . 3 max 47 min Report 0.3 lists the specification requirements for ASTM D 6751. Glucosinolates are compounds that cause the ‘hot’ flavor of condiment mustards and similar products such as horseradish. calcium.050 max 1.3 ASTM D 6751 Specification Requirements (ASTM. These elements will contribute to higher emissions or interfere with the operation of the engine’s emissions control equipment by plugging the Table 7.50 max 0. especially if pour point depressant additives are used. For example. combined Calcium and magnesium.2 max 130 min 0. 2 diesel fuels. Canola is a variety of rapeseed that was engineered by classical selective breeding techniques to reduce both erucic acid and glucosinolates levels. Table 7. Currently. it is a ‘double-zero’ variety that is low in erucic acid and glucosinolates. 2005). While the oil most commonly used for biodiesel in Europe is commonly called rapeseed.050 max 0. sodium and potassium.020 max No.9–6.240 0. This is thought to be due to B100 behaving like a pure substance because of its limited number of constituent compounds (Shrestha et al. combined Oxidation stability Sulfur Cold soak filterability Test method D 93 EN 14110 D 93 D 2709 D 445 D 874 D 130 D 613 D 2500 D 4539 D 664 D 6584 D 6584 D 4951 D 1160 EN 14538 EN 14538 EN 14112 D 5453 Annex 4. low acid) in North America. 2008) Property Flash point (closed cup) Alcohol control: One of the following must be met: a) Methanol content b) Flash point Water and sediment Kinematic viscosity at 40 C Sulfated ash Copper strip corrosion Cetane number Cloud point Carbon residue Acid number Free glycerin Total glycerin Phosphorus content Distillation temperature. 1 or No.020 0. 90 % recovered Sodium and potassium. as set by the American Society for Testing and Materials. even though these elements are not found in the methyl ester molecules.001 max 360 max 5 max 5 max 3 min 15 ppm for S15 grade. phosphorus. Many of the properties of D6751 are determined by the levels of contaminants in the biodiesel. limits are prescribed for sulfur.. magnesium. the additives are more effective with blends than with B100. the cloud point temperature is much lower (Wang and Van Gerpen.0 0. and is thus similar to what is called canola (Canadian oil.Biodiesel from Vegetable Oils 149 When branched-chain alcohols are used.1 Limits 93 min 0. The operability temperature of biodiesel can be lowered by blending with No. 2008). The mustard oils and the variety of rapeseed oil listed in Table 7.2 contain significant amounts of erucic acid as well as glucosinolates. atmospheric equivalent temperature.

also has a high cetane number (Knothe. the high-erucic acid rapeseed. but also an induction period in an accelerated oxidative stability test of 6 h. For example. However. the allowable total glycerin in biodiesel that meets ASTM D6751 is 0.24 % in ASTM D 6751 and 0. which effectively keeps low-cost soybean oil-based biodiesel from competing in Europe. EN 14214 includes a minimum requirement for the ester content (96.8. commercial fuel purchasers are including a BQ-9000 certification requirement in their contracts. which is similar to ASTM D 6751 but with a few differences. Increasingly. The glycerol portion of each of these glycerides is summed to give the bound glycerin value.5 %. The completeness of the transesterification reaction is determined by measuring the amounts of unreacted and partially reacted oil in the form of tri-. despite having a high level of longer chain fatty acids. the cloud point for tallow biodiesel is 15. and requires only a 3 h induction period. and triglycerides. which is subsequently added to the measured value of the free glycerin to give the total glycerin. in order to meet the ASTM specification. and monoglycerides. viscosity. the National Biodiesel Board has initiated a quality program called BQ-9000. Another issue associated with the presence of long-chain fatty esters is that the 90 % distillation temperature and viscosity may be too high to meet the ASTM D 6751 specification (ASTM. or with water-less processes (see below). The measures used to accomplish this purification will be described later in the chapter. the methanol content of the biodiesel is controlled by limiting the flash point. the 90 % distillation temperature for higherucic acid content rapeseed biodiesel is 400  C.24 %. EN 14214 also places an iodine value limit of 120. while the specification limit is 360  C. the reaction must succeed in removing almost 98 % of the glycerin that was originally present in the oil. which normally is very high for biodiesel. These contaminants are removed by either washing the fuel with water. unless it is blended with low iodine value and more saturated feedstocks such as palm oil or animal fat. To help ensure quality in the North American biodiesel market. which is well above the values for the vegetable oils. di-. as exemplified here by rapeseed that has a high erucic acid content. Companies that receive this certification are allowed to use it in their marketing approaches. which is a more saturated feedstock with 40 % saturated fats. EN14214 places not only a limit of 12 % on the content of linolenic acid (a fatty acid that is particularly prone to oxidation). The initial value of the total glycerin level in most common vegetable oils is about 10. As a result. and pour point for several biodiesel fuels. that is based on the ISO 9000 quality system. the documentation of their procedures. In particular.5 %) and individual maximum levels for the mono-. 2003). In contrast. can be lowered to unsafe levels if residual amounts of methanol are present. 2005b). Notably. and an audit to verify their reporting. The program consists of validating a plant’s compliance with ASTM specifications. Longer chain lengths. cloud point. Saturated feedstocks produce biodiesel with higher cetane numbers than unsaturated feedstocks. The flash point.4 shows the cetane number. Among the most important differences between these two standards. has a cetane number of 61. In addition. di-. Table 7. . ASTM D 6751 provides no limit on linolenic acid content. It can be seen that tallow. Both specifications limit the level of total glycerin remaining in the fuel to approximately the same value (0.6  C. The biodiesel specification in Europe is EN14214 (CEN.6 and 56. while the less-saturated vegetable oils have cetane numbers between 49. 2008).150 Diesel from Biomass particulate trap or the exhaust catalyst.25 % in EN 14214). is characterized by a low pour point because the erucic acid is monounsaturated.

7. Water exacerbates this problem by promoting hydrolysis of the oil. and the unsaponifiable fraction of the oil.0 cSt.8 57. with contents of 0. and hydrocarbons.1 %) of up to 5–6 % FFA can usually be processed by simply adjusting the catalyst amount so that. (1994) Peterson et al. Table 7.5 shows typical ranges for FFAs found in common biodiesel feedstocks.0 cSt given in ASTM D 6751.6 À1 0 Pour point ( C) À3. the water content affects the reaction by facilitating the hydrolysis of the oil to create free fatty acids. the extent of saturation of the fatty acids which compose the triglycerides. tocopherols. However.0–1. EN14214. this determines whether heating is required in all of the process tanks so as to prevent gelling of the fuel. The actual steps involved depend on the characteristics of the feedstock oil. (1994) The viscosity of rapeseed oil is 5. there is still sufficient catalyst to enable the reaction to reach equilibrium (Van Gerpen. The latter fraction denotes the portion of the oil that cannot be converted to methyl esters.9 12.6 cSt which is within the allowable limit of 6. and glycerin. 2005). Dry feedstocks (H2O content 0.4 Properties of biodiesel Oil or fat Methyl soybean Methyl tallow Methyl canola Methyl high-erucic rapeseed Cetane number 49. so that biodiesel from high-erucic acid rapeseed could not be used in Europe. Feedstocks with higher water contents should first be dehydrated before being used to produce biodiesel.5 %. which subsequently leads to a saponification reaction with the alkaline catalyst to make soap.1 % maximum being desirable.Biodiesel from Vegetable Oils Table 7. In turn.75 5. Levels above this amount indicate that the oil has been contaminated with foreign material which would need to be removed before the oil is made into biodiesel. The primary parameter used to characterize oil quality is the free fatty acid (FFA) content. . the European specification.65 Cloud point ( C) À1. which constitute the biodiesel. the FFAs complicate the alkaline-catalyzed transesterification process by reacting with the catalyst to produce soap. forming additional FFAs. when the FFAs have been converted to soap.99 4. As mentioned above.18 4.8 Viscosity (cSt) 4.9 61. The degree of saturation of the oil does not significantly affect either the rate or final extent of the reaction.8 À6 À15 Reference 151 Yahya and Marley (1994) Yahya and Marley (1994) Peterson et al. As a result. the specification in North America. The important properties of the feedstock oil are the free fatty acid content.5 Biodiesel Production The production process for biodiesel generally involves the transesterification of an oil or fat with methanol to produce methyl esters. but it does impact on the low-temperature properties of the intermediate and final products of the reaction. This fraction typically includes sterols. limits the maximum viscosity to 5. the content of which rarely exceeds 1. water contents should be lower than 0.6 61. On the other hand.5 %.1 15. the water content.

most producers add 60 % to 200 % excess alcohol.. a 20 : 1 molar ratio of methanol to FFA is required.7 2–7 5–30 40–100 Above a level of 5–6 %. along with sulfuric acid catalyst equal to 5 % of the FFAweight (Canakci and Van Gerpen.5 Free fatty acid (FFA) contents of common biodiesel feedstocks.(Van Gerpen et al. or with a disk centrifuge. and (ii) the glycerin stream. while the products are divided in the separator into two streams: (i) the methyl esters stream.5 %. The oil. and catalyst occurs in the reactor. Coalescence membrane technology has also been used to achieve this (Dube et al. methanol.05 0. such as the use of ultrasonic mixing and cosolvents (see below). when the FFA level has been reduced to less than 0. The FFAs can then be . and catalyst are most commonly mixed in continuously-stirred tank reactors (CSTRs) until the reaction has reached equilibrium.152 Diesel from Biomass Table 7. 2001).3–0. An alternative process involves the vacuum stripping of FFAs from the oil. The reaction of the oil. Figure 7. In either case. In order to drive the chemical reaction to greater oil-to-biodiesel conversion rates. The reaction rate tends to be limited by the low solubility of methanol in the oil. 2007). The glycerin stream passes through an acid addition process.. 2006) Oil or fat Refined vegetable oils Crude vegetable oils Restaurant waste grease Animal fats Trap grease FFA content (%) <0. 1945). The most common approach is to use sulfuric acid to catalyze the esterification of the FFAs with methanol to methyl esters (Keim. 7. according to the following reaction: FFA þ methanol ! methyl ester þ water In order to achieve a high level of conversion of the FFAs. the oil can be processed in the same manner as would occur for a feedstock that has a naturally low FFA content. with 100 % being a common value (Van Gerpen.6 Transesterification As described earlier. Separation of the glycerin and biodiesel can be accomplished using a settling tank with a 1–4 h residence time. which neutralizes the catalyst and breaks the soap into FFAs and salt. 2005). the transesterification process is conducted by reacting the feedstock oil with an alcohol in the presence of a catalyst. some pretreatment is needed to remove the FFAs or to convert them to methyl esters. The catalyst is dissolved in the methanol before adding to the oil. alcohol. and then treating the more concentrated FFA stream with acid-catalyzed esterification. The low-FFA oil stream can subsequently be directed to a conventional alkalicatalyzed transesterification. this minimizes the possibility that a concentrated catalyst might come into content with the oil and thus increase soap production. Several strategies have been developed to accelerate the reaction.1 shows a schematic diagram of a complete biodiesel plant.

as the first reactor approaches equilibrium. additional methanol volumes (60–200 %) are added as reactants and glycerin is removed at an intermediate stage of the reaction. an equilibrium that corresponds to a more complete conversion of the oil to methyl ester is achieved in the second reactor. Reprinted from Van Gerpen 2005. or converted to methyl esters via an acid esterification process. a portion of these materials is lost with the glycerin. In this particular design. 1942. The methanol can be evaporated from the glycerin and recycled. As shown in Figure 7. After separation.Biodiesel from Vegetable Oils Oil Methanol Catalyst Finished biodiesel 153 Dryer Methyl esters Reactor Separator Neutralization and methanol removal Acid Water washing Acid Glycerin (50%) Acidulation and seperation Wet methanol Water Gray water Free fatty acids Methanol / water rectification Methanol removal Wet methanol Methanol storage Water Crude glycerin (85%) Figure 7. Some plants use a sequence of two reactors. as the catalyst and alcohol are more soluble in the glycerin than are the methyl esters. it must be purified with a distillation column before it can be reused. The biodiesel fraction can also be neutralized and any residual methanol removed. In most cases. Bradshaw and Meuly. each stream must be purified and the excess methanol recovered.1.2. As the recovered methanol typically contains water. Although not shown in Figure 7.2. Oil. the glycerin that has been produced to that point is removed. Consequently. methanol. In order to cause the transesterification reaction (Scheme 7. as shown in Figure 7. and a catalyst are combined in the reactor. However. with glycerin separation occurring after each step. a common approach to ensure that complete reaction has occurred is to use at least two CSTRs in series (Bradshaw and Meuly.1) to favor a more complete reaction. it is necessary to add additional reactants and remove any inhibitory product. with permission from Elsevier separated and sold as is. and the methyl esters and glycerin separated following the reaction. this neutralization step is followed by a water wash and a drying process to produce fuel that complies with specification ASTM D 6751. The justification for this added complexity is Le Chatelier’s principle for chemical equilibrium.1 Schematic of biodiesel production plant. and additional amounts must be . 1944) with a glycerin separation process following each reactor.

. ultrasonic agitation. this is addressed through vigorous agitation of the reaction mixture.154 Diesel from Biomass Figure 7. 2006). the reaction rate is limited by the solubility of the alcohol in the oil. thereby increasing the surface area available for reaction. The final reaction state after the second CSTR is more complete than is obtained from a single reaction step. In most cases. Stavarache et al.2 Two continuously stirred tank reactors in series. this has been shown to increase the reaction rate (Noureddini and Zhu. The split of alcohol and catalyst mixture between the reactors varies between 70 : 30 and 90 : 10. and then adding more methanol and catalyst. with 40 kHz giving a faster reaction rate but 28 kHz giving higher yields. On the other hand. was observed to give a good quality reaction in 10 min at 60  C (Harvey et al. 2005. (2003. by permission of Biodiesel Basics added in the second reactor. Interestingly. Collucci et al. Reaction times at 36  C were observed to be 10 to 40 min. Fatty acids in the 1 and 3 positions of the triglyceride reacted faster. 2003). with glycerin removal after each step. 2003. (2007) noted that saturated and unsaturated fatty acids had different reaction rates. . and the use of cosolvents (Harvey et al. The system of two CSTRs still requires excess alcohol well above the stoichiometric amount. Ultrasonic irradiation can also be used to provide the mixing needed to produce the emulsion of alcohol and oil required to achieve a fast reaction rate. an oscillatory flow reactor based on tubes containing orifice plate baffles. (2005) measured reaction rates that were three. Stavarache et al. Stavarache et al. 2001). 2007) reported that methanol cavitates when exposed to ultrasonic excitation and disperses as ‘nanodroplets’ in the oil. (2005) found that higher frequencies were ineffective. 2006. indeed.. For example. Other approaches to address this mass transfer limitation involve oscillatory flow... The two CSTRs are arranged in a series configuration. with most of the reaction occurring in the first 3–10 min. The reaction equilibrium can be modified by removing glycerin after the first CSTR. Early in the transesterification process. Colucci et al. Saka and Kusdiana. 2006. Likewise. 2005. By using such a system. depending on the producer (Van Gerpen et fivefold higher than with mechanical agitation. Reproduced from Van Gerpen et al.. Tests were conducted at both 28 and 40 kHz. with the flow pumped back and forth through the orifice plants. 1997). supercritical conditions.

have investigated the effect of cosolvents on the transesterification process. 2000c).3.. 2000b. 2000a. The process. 1996. tetrahydrofuran (THF). and shown that cosolvent addition causes the oil. Although cosolvent systems overcome the rate-limiting issue of methanol’s solubility in vegetable oil. involves addition of the cosolvent. 2002 since these locations are the most common locations occupied by saturated fatty acids in triglycerides. Reprinted with permission from Boocock et al. 1998. which is shown schematically in Figure 7.Biodiesel from Vegetable Oils 155 Figure 7. This was an important observation. Singh et al. Boocock and coworkers. ketones. the saturated fats react faster. to the methanol and oil. as industrial processes derived from it represent a cost-competitive way of overcoming the limitation imposed by the limited solubility of alcohol in oil (Boocock et al. The glycerin produced later in the reaction also remains in solution. alcohol and catalyst to form a single phase. THF was selected as the cosolvent since it had a similar boiling temperature as methanol. (2007) found that high levels of ultrasonic energy input for extended times led to a reduced yield. at the University of Toronto. this phenomenon being attributed to cracking and oxidation of the methyl esters into aldehydes. they require a greater excess of methanol and more energy input to recover and recirculate the cosolvent..3 Schematic of cosolvent system for biodiesel production. . which results in a single-phase mixture. and shorter-chain organic molecules.

2007). when the biodiesel emerges from the glycerin separation process it still contains traces of free glycerin. In this particular system. Such supercritical processing also simplifies the biodiesel purification process. two alternatives to water washing. The biodiesel and water are allowed to separate in a decanter.09 MPa (Wang and Van Gerpen. either by direct acid addition or by adding acid to the wash water. and soap. Water washing is the most commonly employed technique to do this. the soap splits into a FFA (which is soluble in the biodiesel) and a salt (which is easily washed out). In the absence of such pH adjustment. the biodiesel is dried and tested to assess its final quality. because no soap is produced and there is no catalyst residue in the resulting methyl esters. 2001. however. raised safety concerns in the plant. Experimental data have demonstrated the need for high methanol : oil molar ratios such as 42 : 1. The first option is to use a solid absorbent such as magnesium silicate (sold commercially as Magnesol) (Bertram et al. The biodiesel is mixed with additional pure water (deionized to prevent the transfer of calcium and magnesium to the biodiesel) for a second wash. a drawback here was that greater excesses of methanol were required to offset the dilution effect of the cosolvent. although a proposed two-step process might reduce this to requirement to ratios in the range of 10 : 1 to 15 : 1 (D’Ippolito et al. . however. An additional point was that the toxicity of the proposed cosolvent. both compounds could be recovered simultaneously at the end of the reaction and returned to the main process stream. this ratio remains considerably higher than the value of 6 : 1 used conventionally for alkali-catalyzed transesterification reactions. Notably. 7. At low pH. The water that emerges from this second decanter is re-used for the first washing step. This process is characterized by short reaction times at ambient temperatures (7 min at 23  C). 2001). water is mixed with biodiesel in counterflow in packed columns. which can be added to the biodiesel when the methanol has been removed.156 Diesel from Biomass as a result.0. biodiesel is mixed with recycled water that has already been used once to wash biodiesel. thus eliminating mass transfer limitations and ensuring that fast reaction rates are achieved even in the absence of any catalyst (Saka and Kusdiana.5–5. and to which acid may be added to neutralize the catalyst and split any soaps.. In order for the fuel to meet ASTM specifications. This acid treatment is effective because soap is the contaminant that is the most difficult to remove. these contaminants must be reduced to low levels. Kusdiana and Saka. methanol.7 Biodiesel Purification As discussed previously. When these operations have been completed.. If the pH of the biodiesel is lowered to 4. after which the spent wash water is sent to wastewater treatment. typically 100–200 % of the biodiesel volume. A typical system for combining acid addition with a two-stage counterflow washing process is shown in Figure 7. (2007). the biodiesel and water are subsequently separated in a second decanter. THF. 2005). (2005) and He et al. The critical temperature and pressure of methanol are 512 K and 8. in this way the soap and other contaminants are transferred to the water. There are. methanol acts as a powerful solvent. catalyst. then the amount of water needed to achieve adequate purification can be kept to 5 % of the biodiesel amount. 2002).4. large volumes of water are required to remove the soap. as reported by Vera et al. At temperatures above this point. In one approach.

some of the soap may precipitate as a gelatinous material that may plug the filters and screens. which acts by removing the soap. and traces of methanol. Counterflow washing systems use water very efficiently by using the same water for two washes. When magnesium silicate or ion exchange resins are used to purify biodiesel. The system shown uses acid for the first wash to split any soaps. may fail the ASTM specification for acid value (ASTM. so as to produce fuel with a bright. if the fuel is water-washed. so that the total glycerin level of the fuel can also be lowered. catalyst. Reprinted from Van Gerpen et al.Biodiesel from Vegetable Oils water biodiesel acid water 157 Washed biodiesel Gray water Gray water Figure 7.4 Continuous counterflow water washing system. and collect at the inlet to the ion-exchange resin. A second option is to use an ion-exchange resin (e. Rohm and Haas). This water can be removed by heating the fuel under pressure and then spraying it into a vacuum flash chamber. catalyst. It has been observed that. In practice. by permission of Biodiesel Basics The magnesium silicate acts by absorbing any polar contaminants such as free glycerin. when methanol is removed. These resins can often be used while methanol is still present in the biodiesel. however. Recent concerns about sterol glucosides have motivated many producers to install the capability to conduct cold filtering of their product before it leaves . it has been noted that even when biodiesel meets all of the requirements of the ASTM specification. as is common for high FFA feedstocks. 2008). BD10Dry. this is an important advantage because methanol acts as a cosolvent for the biodiesel and soap. Ion-exchange resins do not actually remove soap. near-transparent appearance.. rather. there may be small suspended water droplets that give the fuel a cloudy appearance. An added benefit of this method is that the magnesium silicate also absorbs some of the monoglycerides. These have been traced to small amounts of compounds known as sterol glucosides.g. so that when the methanol is vaporized no soap precipitation will occur. 2006. there are occasional incidences of solid precipitates found in the fuel that can cause filter plugging. and free glycerin. Ion-exchange resins allow the soap to be removed first. Fuels with soap levels above 2500 ppm. the residual water level should be very low. soap. they convert the soap into FFAs that pass through the resin bed and contaminate the fuel..

the conversion of vegetable oils to biodiesel is an important step. Different compounds are possible depending on the functional group attached at the location designated as R. However. . The consequences of these phenomena is filter plugging. this creates a molecule referred to as the ‘nonacylated’ form of the sterol glucoside. The structure of a typical sterol glucoside is shown in Figure 7.cyberlipid. 300 ml of fuel is cooled to 4. 2008). as described earlier.6). A test procedure developed to replicate the cold-filtering process is currently used to determine whether fuels are likely to cause filter-plugging problems (see Figure 7. such as saturated mono. and then reheated to 20–22 C for 2.158 Diesel from Biomass R = H or C15H31CO CH2OR O O OH OH OH Figure 7.and diglycerides and saturated methyl esters.7 mm glass fiber filter is recorded. the precipitate complexes are generally of a greater mass than if they had been generated solely by the precipitation of sterol glucosides. it may be found to contain a sterol glucoside precipitate.8 Perspective Vegetable oils have been demonstrated to constitute suitable substitutes for diesel fuels. In nature. Biodiesel production practices are today well established.4  C for 16 h. the functional group is a 15-carbon fatty acid chain. Thus. when the fuel has been produced and stored for a few days. as alkyl esters do not generate the excessive deposits and wear observed when simple vegetable oils are used in diesel engines. one unintentional consequence is the stripping of the 15-carbon fatty acid chain and its replacement with a hydrogen atom.5 Sterol glucoside molecule (http://www. In this test. which is insoluble in the biodiesel. (2007) have proposed a mechanism to explain the problems associated with the presence of plant sterols in biodiesel. especially at lower temperatures. Lee et the plant.5 h. The time required to filter the entire fuel sample through a 0. and the molecule is soluble in vegetable oil. As a result. Filtration times above 360 s correspond to fuel that is likely to plug the fuel filter on an engine (ASTM. present as dispersed fine solid particles with very high melting points. During the biodiesel production process. and when the fuel meets the ASTM specifications (or other similarly stringent specifications) no specific problem is observed when this renewable fuel is used to power conventional diesel engines. A complicating factor here is that these small particles apparently act as nucleation sites for the crystallization of other compounds. 7. Water may also be a complicating factor in the formation of large particle complexes.

This difference focuses the energy industry’s attention on a dichotomy that frequently exists with biomass fuels – whether the savings from reduced transportation costs associated with distributed plants located near feedstock sources can offset the economies of scale of very large plants. Although today. 2007. low-cost production facilities. 1991. because renewable diesel is produced using processes that are common in petroleum refining. most biodiesel is produced in decentralized plants that are small compared to petroleum refineries.. hose. However. In addition. 2006). it has been criticized for slight increases (2–10 %. and wire with appropriate sealant File clamp jaws and handle where wire attaches to bare metal Clamp To Vacuum Pump Wire to Ground Vacuum Tubing Safety Flask Receiving Flask Figure 7. as the final product is also similar to petroleum-based diesel fuel.. Biodiesel is produced using processes not currently found in refineries.. while the reaction conditions are easily provided in small. research is under way to locate new sources of oil (Altiparmak et al. renewable diesel will be favored by the existing petroleum industry. In warm climates. Foidl et al. Reprinted with permission from ASTM 2008 The distinction between renewable diesel and biodiesel will become increasingly important.. this is unlikely to affect the competitiveness of biodiesel with petroleum diesel fuel unless the cost of feedstock can be reduced. however. Although biodiesel reduces the exhaust emissions of CO. 2007). 1996). The exhaust after-treatment devices will reduce emissions to such low levels – regardless of the fuel – that differences between fuels may not be significant or useful as a selling point. it needs to be domesticated.6 Apparatus used for the cold filterability test. Sahoo et al. depending on blend level) in the emission of oxides of nitrogen. which will keep their cost high. which includes not only identifying pesticide and fertilizer requirements but also taking measures to address disease issues that inevitably will arise from high-density . The effect of biodiesel on emissions is likely to become moot when current emissions regulations are fully implemented in 2010.. unburned HC and PM. Approximately 80 % of the cost of producing biodiesel is the cost of the feedstock oil (Van Gerpen et al.Biodiesel from Vegetable Oils Funnel Vacuum Tubing 159 Laboratory Ground (Typical) Insure seal between tube. jatropha is seen as a potential new source of oil as it can be grown on arid lands not suitable for crop production (Ouedraogo et al. Whilst processing techniques will improve and become more efficient over time. some of these sources are currently used as food. Thus.

K. V.. Durability and Low Temperature Evaluation of Sunflower Oil as a Diesel Fuel Technol. Baranescu. Biodiesel Standard EN14214. A. Cheng. 7: 297–318. G.B. The extent of its impact will depend on whether adequate supplies of low-cost feedstocks can be developed. West Conshohocken. D.R. G. Awarded 9 April.C.L. Fuel Process. African J. Preparation of detergents. Mueller. Kyoto University International Symposium on Post-Petrofuels in the 21st Century – Prospects in the Future of Biomass Energy. Awarded 16 August. US Patent 6.S. Code of Federal Regulations. and J. G. 1996. C. Brussels.B. B. Fast formation of high-purity methyl esters from vegetables oils.368.C. 2006. Barberio. Chemical composition and insecticidal properties of the underutilized Jatropha Curcas seed oil.J. Biodiesel fuel from waste fats and oils: A process for converting fatty acids and triglycerides. August 2004. 1981.K. 2002. Biotechnol. invasive species contamination and high water consumption have yet to be overcome (Nagle and Lemke. Vispute and G. Boocock.. Mao. U. D. Guru. T. Part 86: Control of Emissions from New and In-use Highway Vehicles and Engines. 1982. pp. PA.B. Am. 32: 4313 (1938). and C. Algae is seen as another opportunity for large amounts of new oil. Engine Res. S. Biomass Bioenergy 11 (1): 43–50. A. http://www. and W. M. Adedire.360. D.G. Belgium. 2002c. U. Altiparmak.gpoaccess.844 Awarded 24 October.P. and J. (Translation from Knothe. 1944.B. Fargo. Biodiesel has the potential to provide a significant portion of the world’s future supply of fuel for diesel engines. 86: 1679–1693.. Boocock. Abstr. 1944. U. Meuly. and A. Sidi. D 6751-08 Standard Specification for Biodiesel Fuel Blend Stock (B100) for Middle Distillate Fuels in Annual Book of ASTM Standards. 44 (6): 1429–1436. Int. and W.G. 2002. Awarded 22 July.J. Dibenedetto and G. Alternative Fuel Properties of Tall Oil Fatty Acid Methyl Ester-Diesel Fuel Blends. Performance and Emissions Characteristics of a Naturally Aspirated Diesel Engine with Vegetable Oil Fuels.J. SAE Paper 810262. A. Canada. CEN 2008. Fast one-phase oil-rich processes for the preparation of vegetable oil methyl esters.648. JAOCS 75 (9): 1167–1172. . Biodiesel production from oils and fats with high free fatty acids. Lee and S. Agric. 2005. Eng.B. Chem. D. Process for production of fatty acid methyl esters from fatty acid triglycerides. 2008. Carlson. Konar. but the problems of high cost.399. Patent 6. Canakci.. Van Gerpen 2001. ASAE. Title 40. Aresta et al. ASTM. Patent 2. Adsorbent filtration system for treating used cooking oil or fat in frying operations. 2007. References Adebowale.W. Huber. Technol.S.Kauffman. D.S. ND. Humke. A.877 (31 August. Soc. Koca and M. 312–328. J. Boocock. ASTM International. M. K.B. Patent 2.867.S. U.G. European Committee for Standardization (CEN). Patent 6. 1942. 3–4 September. 2000. Bradshaw..271.. Procedure for the transformation of vegetable oils for their uses as fuels.G. Barsic. Buligan. D.S. 5 (10): 901–906. R. Chavanne. Konar. 2002b. 1998.B. Boocock. 2002. Biores. 1990.O. S. Investigations of the Impact of Biodiesel Fueling on NOx Emissions Using an Optical Direct Injection Diesel Engine. 98: 241–246. 2005).619. Montreal.160 Diesel from Biomass cultivation. C. Abrams and J.712. Proceedings of the International Conference on Plant and Vegetable Oils as Fuels. A.html. Boocock. Meuly. ChemSusChem 1 (5): 397–400.G. 2000a. N. Performance.. Trans. Bertram. Aresta. Mao and H. 2008. Utilization of macro-algae for enhanced CO2 fixation and biofuels production: Development of a computing software for an LCA study. Bradshaw. Green gasoline by catalytic fast pyrolysis of solid biomass derived compounds. Lusco. 1937). Process of making pure soaps. V. Upatnieks and C. 2005c) Belgian Patent 422.642. Keskin. T.A. CFR 2008. 2006. Single phase process for production of fatty acid methyl esters from mixtures of triglycerides and fatty acids.O.

Appl. Engelman and D. 2001. New York.Y.. 1945. Catal. J. Proceedings of the International Conference on Plant and Vegetable Oils as Fuels. R. Harvey. http://www. Knothe. O’Connor and A. C. 2005. Biodiesel Magazine.P. 2007. ASAE. Barrett and J. D. Catal. J. 279–286. 2003.W. R. E.E. Van Gerpen and J. and N. ND. 98: 639–647. C. J. 2005b. Kinetics of transesterification in rapeseed oil to biodiesel fuel as treated in supercritical methanol. M. JAOCS 82 (7): 525–530. Seliger. Technol.. Cetane Numbers – Heat of Combustion – Why Vegetable Oils and Their Derivatives are Suitable as a Diesel Fuel. pp. pp. Science 308: 1446–1450.R. Poppe. A: General 199: 147–190.E. 2nd edn.J Barsic.. Goering. SAE 2007-01-4042. Borrero and F. A: General 329: 120–129.A. Treating Fats and Fatty Oils. 78: 338–341. Dumesic. J.W. Comparison of emissions and mutagenicity from biodiesel. Zhu. pp. Fuel 80: 693–698.S. Heywood.conocophillips. Knothe. 2000. 1945. Furimsky. April.. G. Mittelbach and S.. S. L.A. Transesterification kinetics of soybean oil for production of biodiesel in supercritical methanol. 1981. A. R. Kiannejad.I. Keim. C. Kusdiana. Fargo. J.S. Powers and T.conocophillips.. Chem. 1912. Technol. Mackley and T. Kinetics of transesterification of soybean oil. ND. as a source for the production of biofuel in Nicaragua.J. in The Biodiesel Handbook. Chheda. pp. M. noncatalytic. G. M.. C. A. Foidl.A. Bunger. ConocoPhillips. Corma. Liu. Fuls. 76–82. H. Krahl. P. Van Gerpen and J. H. 2008). Single and Multi Cylinder Diesel Engine Tests with Vegetable Oil Emulsions.B.M. Production of liquid alkanes by aqueous-phase processing of biomass-derived carbohydrates. Munack. Nazha and F. ASAE. http://www. Biodiesel from an alkaline transesterification reaction of soybean oil using ultrasonic mixing. Wang and S. Hackel. Iturria.J. Champaign. A. Huber. 2005. Engineering 93: 395–406. IL. S. G. 1982. Champaign.C.A.L.W. Haines. O. M. 58 (1): 77–82.B. Zhu. Biores. W.htm (Accessed 27 April.. Crookes. Saka. SAE Paper 810955. and S. Evaluation of Soybean Oil-Aqueous Ethanol Microemulsions for Diesel Engines. Comparative Combustion Studies on Various Plant Oil Esters and the Long Term Effects of an Ethyl Ester on a Compression Ignition Engine.383. 2007. Analysis of a two-step. 2007. Schroder and J. 2–4 August. Appl. supercritical biodiesel production process with heat recovery. The Diesel oil engine. 2005a.htm (Accessed 27 April. Process intensification of biodiesel production using a continuous oscillatory flow reactor. Fishinger. 2007. He. 312–328. Mass.. Biores.601. Biodiesel production using a membrane reactor.C. M. news_releases/2007news/04-16-2007. Technol. D’Ippolito. News Release: ConocoPhillips and Tyson Foods Announce Strategic Alliance to Produce Next Generation Renewable Diesel Fuel. Noureddini.H. H. Hawkins. 2006news/12-19-2006. Organic Chemistry. August 2004. Lee. 1997. AOCS Press. Biotechnol. Guenther.A. Service Trial of Waste Vegetable Oil as a Diesel Fuel Supplement. Lexington. Dube. Knothe. SAE 811215.. News Release: ConocoPhillips Begins Production of Renewable Diesel Fuel at Whitegate Refinery in Cork. Vera. and J. M. Ireland. Pfalzgraf.M.W. Campion. Jatropha curcas L. G. 2007. Patent 2. I. G. AOCS Press.C. A.E. Humke. T. 1982. G. and D.A.. Krahl (eds). Sanchez. GTL and diesel fuel. Catalytic hydrodeoxygenation. Ruschel. SAE Paper 922230. A. U. vegetable oil. 4–16. J.K.Biodiesel from Vegetable Oils 161 Colucci. 1970. Knothe. Heath and Company. Fargo.R. E. Diesel.L.. .N. 2007. 1982. Proceedings of the International Conference on Plant and Vegetable Oils as Fuels. 1982. D. Pieck and C. in The Biodiesel Handbook. Pryde. Yori.W. Krahl (eds).. Huber. Energy and Fuels 21: 339–346.. 1981. Alape. The role of sterol glucosides on filter plugging. N. 1996. Linstromberg. The History of Vegetable Oil-Base Diesel Fuels. J. Processing biomass in conventional oil refineries: production of high quality diesel by hydrotreating vegetable oils in heavy vacuum oil mixtures. M. Foidl. G. JAOCS 84: 399–404. JAOCS 74 (11): 1457–1463. Schwab and E. 2007. 1988. 1992. Performance and Emissions Characteristics of a Naturally Aspirated Diesel Engine with Vegetable Oil Fuels – (part 2). J. ConocoPhillips. Internal Combustion Engine Fundamentals. G. Sun. 2008). 2006. IL.A. Tremblay and J. G. McGraw-Hill. Awarded 28 August.

L. Organosulfonic acid-functionalized mesoporous silicas for the esterification of fatty acid. Ultrason. Sahoo. L. Shrestha.. Peterson. L. M. 1992.. R. H. I.M.K. pp. Mbaraka. Falcon. Proceedings of the Institution of Mechanical Engineers.K. Rantanen. World Oilseeds – Chemistry. Biodiesel development from high acid value polanga seed oil and performance evaluation in CI engine. Either Straight or in Blends. R.N. Vegetable oil as a diesel fuel: status and research priorities. The effect of minor components on cloud point and filterability. I.K.C. D. 198–208.H.. Kadam (eds). I Mech E 210: 47–53. D. Maeda. Fernando and R. Biodiesel fuel from rapeseed oil as prepared in supercritical methanol. C. 2003. Linnaila. Stumborg. Journal of Power and Energy. Masjuki and A. Aakko and T. Ouedraogo. Appl. Singh.A.. Fatty acids methyl esters from vegetable oil by means of ultrasonic energy. Warrendale. NExBTL – Biodiesel fuel of the second generation. Pischinger. Maeda.H. U. Energy and Fuels 21: 1161–1164. 2008. 1990. and Shanks. Biotechnol. 1986. C. and A. 13: 401–407.M. Biodiesel Emissions Data from Series 60 DDC Engines. Wong. 1982. Radu. S. J. P.D. R. Monnier. 29: 1413–1422.. Fuel 80: 225–231.. Foster and G. Catal. Shanks.G. Poppe. S. PA. Biodiesel Magazine. Presented at the 1995 American Public Transit Association Bus Operations and Technology Conference. Chavan. J. Design of multifunctionalized mesoporous silicas for esterification of fatty acid. P. 2000. Technology and Utilization. ASAE Paper 946531. SAE. Lett. Conversion of oils and fats using advanced mesoporous heterogeneous catalysts. 32 (8): 716–717. 2001.. A. Presented at the 1994 ASAE International Winter Meeting.K. Babu and S. C. Diesel engine performance tests using oil from Jatropha curcas L.H. P. 1995. Ultrason. Peterson. Stanislao. Van Gerpen and J. 1997. Pfalzgraf. Tourigny. R. Pestes. ASAE.L.S. Sapuam. Sonochem.K. Howell and J. ASAE Trans.. J. 2–4 August. Adsule and S. Production of methyl ester fuel from microalgae. N. 2006. Agricultural Mechanization in Asia. S. 2006. B. J. Kusdiana. Reece. Soveran.. The use of palm oil as diesel fuel substitute. Sonochem. Vinatoru. 219: 329–336.K. Lemke.. I. B. Maeda. Linden. Kinetics of transesterification of soybean oil. C. Methylesters of Plant Oils as Diesel Fuels.. Ultrasonic versus silent methylation of vegetable oils.. NV. Africa. 1995. Part 1: Regulated Emissions and Performance. D. Characterization and Performance of Eight Fuels from Lipids. and P. ND. Society of Automotive Engineers Paper No. M. Sharp. Sii. Stavarache. 2007.. 2005. C. Mbaraka. A... Harju.N. Thompson.H. Naik. JAOCS 74 (11): 1457–1463. JAOCS 83: 79–91. D. 2005.162 Diesel from Biomass Mbaraka.-Y. December 13–16. V. Zhu. 24–25 (1): 355–361. J. B. Reno. Trans. 2007. Lee. Vinatoru. Processing. and D. Nishimura and Y. 1996. 1991. Hernandez.S.H. Van Nostrand Reinhold. S. Nishimura and Y. The Effect of Biodiesel Fuels on Transient Emissions from Modern Diesel Engines.722. 32. H. M. Hammond and J. Piston ring deposits when using vegetable oil as a fuel. 2000-01-1967. Effectiveness of Cold Flow Additives on Various Biodiesels. Zaki. Testing and Evaluation 12: 61–68. Biochem. Diesel. .J. Saka. Stavarache. R. Dynamometer evaluation and engine wear characteristics of palm oil diesel emulsions. Nagle. and Latin America 22 (4): 25–29. Conversion of vegetable oil to biodiesel using ultrasonic irradiation.K.K. Vinatoru. Stavarache. C. Fuel 86: 448–454. Part A.705. D. 12 (5): 367–372. Ayers and J.N.A.R.L. L. Fargo. Thompson. M. 2007. Jobe.L. M. G. Catal.. Das. E. and D.S. Nishimura and Y. 1984. Noureddini.G. Base-catalyzed fast transesterification of soybean oil using ultrasonication. New York. Schumacher. Lin and B.M. J. Masjuki and A. ASABE 51 (4): 1365–1370.D. Proceedings of the International Conference on Plant and Vegetable Oils as Fuels. 2003. JAOCS 72 (8): 905–909. Atlanta. and J. November. 2005. H.S. Patent 5. GA.N. Chem. 229: 365–373.. Hogan and M. and Shanks. I. M.M. SAE Paper 2005-01-3771. Conversion of biomass feedstock to diesel fuel additive.W. and Their Blends. H. G. Salunkhe. Azlan. S. 1998. J.

Aspects of ultrasonically assisted transesterification of various vegetable oils with methanol. M. 2008. Biodiesel processing and production.H. and J. Rio de Janeiro. Article on CNN Money website.C. 1983. SAE 831222. Z. P. 2007.. 14: 380–386. Unpublished measurements of fatty acid profiles in various plant oils. 2003. Van Gerpen. Tat. Las Vegas. and S. Wormley. Van Gerpen and P.S. Physical and chemical characterization of methyl soy oil and methyl tallow esters as CI engine fuels. Diesel Cars are Coming Back. Building a Successful Biodiesel Business. Peters and B.. University of Idaho. R. S. Presented at the 2007 Agricultural Equipment Technology Conference. J.L. 2005.S. E. D’Ippolito. Wang. Vinatoru. Accessed 9 March. R. Dubuque. . Shanks and G. Soybean oil methyl esters preparation using NaX zeolites loades with KOH as heterogeneous catalyst. J. 2007. Wang. 2008. C. M. Clements. Vegetable Oil or Diesel Fuel – A Flexible Option. 27–30 July. 50: 1123–1128. Fuel Process. Biodiesel Basics. Dumesic. August 14–18. Li. Marley. Van Gerpen. R. Production of Biodiesel by a Two-Step Supercritical Reaction Process with Adsorption Refining. Schroer. P. The speed of sound and isentropic bulk modulus of biodiesel at 21 C from atmospheric pressure to 35 MPa.L. 2007.E. Ziemke. Valdes-Dapena. JAOCS 82 (11): 845–849. Xie.Y.J. Biodiesel: An Alternative Fuel for Compression Ignition Engines. S.. 2003. Long-term Operation of a Turbocharged Diesel Engine on Soybean Oil Fuel Blends. Huang and H. Wang. Van Gerpen. Knothe. W. 1994. SAE 840004.. Van Gerpen.Biodiesel from Vegetable Oils 163 Stavarache. Liu. M. M. Ultrason. C. Parera. 2007.. J. ASAE Paper 036034. Yahya. Vera. 2005. Fuel property effects on injection timing. H. C. Goering.A. KY 11–14 February. ignition timing. Van Gerpen.. Pieck and J. 98: 936–939. ASABE Trans. J. A. J. IA. Technol. and oxides of nitrogen emissions for biodiesel-fueled engines.cnn. Soylu.M.E. 2009.H. Biores. 2007.. 2006. B. P. Peter and J. 2005.S.M. Nishimura and Y. X. Tat. Canakci. American Society of Agricultural Engineering Annual Meeting. ChemSusChem 1 (5): 417–424. J. D. Van Van Gerpen. JAOCS 77: 285–289. Sonochem. FL. Monyem and S. 2005 ASAE Annual Meeting.. Tat and J.R. A. Tat. ASABE Distinguished Lecture Series. Biomass Bioenergy 6: 321–328. 2nd Mercosur Congress on Chemical Engineering. Liquid alkanes with targeted molecular weights from biomass-derived carbohydrates. June 5. NV. http://money.. Maeda. Pruszko. Fuel Property Effects on Biodiesel. 86: 1097–1107.F.J. C. 31. Louisville.. 2000. The production of isopropyl esters and their use in a diesel engine. M. ASAE Paper 056124. Technol. and J. 17–20 July. Tampa. Tractor Design No. M. M.E. Catalyst-free Biodiesel Production at Elevated Temperatures Including Supercritical Conditions: A Review. 2005.E. M.A. K. 1984. West. Suda. Peterson and C.


1 Introduction Seaweeds and microalgae have a long history of cultivation as sources of commercial products (McHugh. Hans Blaschek e and Hideaki Yukawa The contribution of Huesemann. It is hoped that this will lead to a dramatic expansion in the production of transportation biofuels. in particular liquid biofuels for transportation (Huesemann. without competition for arable land that would impact food and feed supplies. these photosynthetic organisms are again a focus of interest as potential sources of biofuels.8 Biofuels from Microalgae and Seaweeds Michael Huesemann.. particularly liquid transportation fuels. Seaweed cultivation has received relatively little attention as a biofuel source in the US. Roesjadi and Metting has been written in the course of their official duties as US government employees and is classified as a US Government Work. Nasib Qureshi. Presently. There have been many recent private sector investments to develop biofuels from microalgae. As energy costs rise. Japan and Korea. including about 2 % of transportation fuels as ethanol and biodiesel derived from corn and soybeans. which is in the public domain in the United States of America. respectively. 2002). 2006). . Biomass to Biofuels: Strategies for Global Industries Edited by Alain Verts. Roesjadi. 2003. 1998). Extensive research and development of so-called ‘next-generation biofuels’ – ethanol from lignocellulosic biomass and biodiesel from unconventional oilseeds and microalgae – is now under way. Pulz and Gross. about 4 % of the US total energy demand is supplied by biomass-derived energy. 1987). They also have been the subject of extensive investigations related to their potential as fuel source since the 1970s (Chynoweth. but was the subject of a major research effort by the DOE from 1978 to 1983 (Bird and Benson. Blaine Metting 8. in part building on a US Department of Energy (DOE) program from 1976 to 1996 which focused on microalgal oil production (Sheehan et al. Increasing fossil fuel prices and greenhouse gas mitigation goals necessitate the development of renewable energy sources. G. and is now the focus of significant interest in Europe. John Benemann and F. 2004).

causing economic. and can be carried out in shallow ponds on hardpan soils. with projected productivities for other microalgae and seaweeds of up to 70 mt haÀ1 yrÀ1 (Hanisak and Samuel. not a highly productive species.. are estimated at about 40 mt haÀ1 yrÀ1 (all productivities are reported in this review as dry weight organic mass) in climatically favored locations. thus avoiding water and nutrient limitations.. using saline or brackish water or. the drawbacks of algal biofuels production must be considered. irrigated sugar cane in the tropics) can approach the productivities reported for algae. although algae do not have an inherently more efficient photosynthetic process and some vascular plants (e. such as flue-gases from fossil fuel-fired power plants. and 10–13 mt haÀ1 yrÀ1 projected for switchgrass or hybrid poplars (Perlack et al. These positive attributes could. 1988. Sheehan et al. 2005.5 g mÀ2 dayÀ1 for an annual average at favorable locations for year-round production. The first advantage is their potentially greater productivity growing in water. 1987. 1988. The second advantage is that algal cultivation does not require arable land. as well as their potential for continuous cultivation at near maximal productivity.1 Comparative average productivities of terrestrial and marine ecosystems with engineered microalgae systems. have driven up food prices. in coastal or theoretically in open-ocean settings. Thus. including high cost and the undeveloped nature of their production processes. Jassby. Commercial biomass productivities for the cyanobacterium Spirulina (Arthrospira platensis). Table 8.g. 2002). favor the largescale production of algal biofuels. for seaweeds. . algae have few nonproductive parts such as roots or stems. this value is generally agreed upon in the scientific community. social and even ethical problems (Rosegrant et al. along with oil prices and other factors. Further. in principle. Microalgae systems require an enriched source of CO2. resulting in a proportionally smaller areal footprint for algal systems. b 100 t haÀ1 yrÀ1 of C fixed into biomass is the approximate theoretical maximum efficiency of photosynthesis. Of course.1). and referred to in the text.) Ecosystem Terrestrial Tropical rain forest Temperate evergreen forest Cultivated land Marine Seaweed beds and reefs Microalgae Microalgae raceway pondsa Achievable photosynthetic yieldsb a Net primary productivity (g C mÀ2 yrÀ1) 1000 600 300 1350 2000 10 000 World average for current commercial Spirulina production of ca.166 Diesel from Biomass Algae have two major advantages over higher plants with respect to biofuels production.. while avoiding competition with food and feed crops that has recently upset the worldwide supply–demand balance and. 9 mt haÀ1 yrÀ1 for corn. 2006). they certainly are among the most productive photosynthetic organisms (Table 8. 5. and can be colocated with such sources. McLaughlin et al. This compares to 3 mt haÀ1 yrÀ1 for soybeans. (After Harlin and Darley. 1998)...

1). Sawayama.2. like other high-temperature thermal processes. and describe the current commercial and engineering approaches to algal biomass production..2 8. and Limitations Combustion and Thermochemical Conversions Microalgae can potentially generate a wide range of biofuels (Figure 8. 48. Hydrogen Hydrogen Biochemical Conversion Gasification Microalgal Biomass Thermochemical Conversion Pyrolysis Liquefaction Hydrocarbon Gas Oil.1 Biofuels from Microalgae: Products. since drying is expensive. while combustion. Adapted from K. Gas. which is about 100-fold that of Dark Fermentation Anaerobic Digestion Photo-Fermentation Biophotolysis Ethanol.1 Overview of algal biomass conversion processes for the generation of biofuels and bioenergy. Hydrogen Ethanol. Processes. destroys the nitrogen fertilizer content of the biomass and generates elevated emissions of NOx. Even solar drying is not without cost. 2005. Algal biomass typically contains 5–10 % N.and macro-algal feedstocks will become competitive with other biofuels or renewable energy sources. The potential for an algal biofuels industry is discussed. In this chapter. However. Liquid fuel production using microalgae. Journal of the Japan Petroleum Institute. 1995. Charcoal Oil Chemical Separation Solvent Extraction Oil Direct Combustion Power Generation Electricity or Power Figure 8. Hydrogen Methane. Kadam. this approach is the least attractive option. harvested algal biomass (typically 80–95 % moisture content) could be dried and combusted to generate electricity (Matsumoto et al. Most simply. 251–259 . we briefly review the history of biofuel research with microalgae and seaweeds. Tsukahara. 2002). and S.Biofuels from Microalgae and Seaweeds 167 Major biological and technical barriers remain to be overcome before fuels from both micro. and the R&D challenges that must be addressed are outlined. 8.

1983a). Thus. Gasification using near-critical water temperature–pressures has been advocated because this requires no drying.168 Diesel from Biomass woody biomass. 30 %) in methane production rates for microalgal biomass harvested from sewage ponds (Chen and Oswald. 1978. These various processes are described in the following sections. Further. 18 MPa). Peng et al. > 400  C) and pressures (i. 2001b. Other microalgae accumulate carbohydrates that can be converted by yeast or bacterial fermentation to ethanol. 8. 2000. water vapor. The anaerobic digestion of microalgal biomass has been successfully demonstrated over the years by a number of investigators (Golueke et al. Another argument against the combustion of algal biomass is that it competes with cheaper woody biomass or coal costing less than US$ 5 GJÀ1.. Sawayama et al. 2004. Miao and Wu. Reported yields of methane were typically around 0. butanol or other liquid fuels. Minowa et al.5 to 3. considering the required high temperatures (i. thus doubling the methane yields by . and this resulted in a significant increase (ca. 2005).2. 1986).. and small amounts (normally <1 %) of hydrogen sulfide and sometimes hydrogen (Gunaseelan.e.. In the case of Spirulina maxima biomass.2 Methane Production by Anaerobic Digestion Anaerobic digestion is the microbial conversion of organic matter to biogas – a mixture of methane. 1995. 1957.. Wu et al. 1997).. produces hydrocarbons that might be refined like petroleum... this is the ratio of biofuel energy output to process energy input) usually ranging from 1. and the nitrogen is converted to recoverable ammonia (Tsukahara and Sawayama. 1999). the more promising options are to recover biofuels from the wet algal biomass directly after harvest. the likely limiting factor accounting for low yields is ammonia inhibition (Samson and LeDuy. 1990. with little regard to composition. CO2. which does not have a strong cell wall. Samson and LeDuy. Finally. Uziel.’ One microalga. The most direct approach is to extract vegetable oils from the biomass. the entire biomass.3 l gÀ1 volatile solids destroyed. Although the thermochemical conversion of microalgal biomass through gasification or pyrolysis to produce synthesis gas or oils has been demonstrated experimentally (Miao et al. including the recalcitrance of some algal species to biodegradation. which is about half of the theoretical maximum based on the biochemical composition of the biomass.. this remains an industrially challenging process to scale up. can be converted to biogas by the well-known methanogenic process of anaerobic digestion.. 1979. so-called ‘green diesel. 2004. Minowa and Sawayama.e. Rigoni-Stern et al. which also suggest a rather low EOR. 1999. Botrycoccus braunii. 2001a.. or converted by hydrocracking into a diesel-like. this problem can be circumvented by adding carbon-rich wastes to the microalgal biomass. 1999. compared to liquid fuels at over US$ 20 GJÀ1. these processes are also limited by the drying requirement and nitrogen loss. However. Eisenberg et al. However. which can then either be readily converted via transesterification to biodiesel. Thermochemical and mechanical pretreatments have been shown to solubilize the biomass by breaking down the cell walls which are resistant to biodegradation. This low yield is due to several factors. and the inhibition of the microbiological conversion process by ammonia released from the biomass. 1998). the fuel products cannot be readily utilized without expensive upgrading processes with rather modest net energy output ratios (EORs.

3 Ethanol and Other Solvent Fermentations There are two alternative processes by which ethanol can be generated from microalgae. 1984. 1998). There are three principal processes by which hydrogen can be generated by microalgae: (i) dark-fermentations...2. including algal biomass produced during wastewater treatment and for the conversion of residuals remaining after oil extraction or fermentation to produce more valuable liquid fuels. 2005). 2007): . glycogen in cyanobacteria. 1997. and (iii) biophotolysis.. 1981. 2004.2. Compared to other microalgal energy conversion processes. and has a high EOR except in colder climates where digester heating is required. but this process also suffers from very low ethanol yields (Akano et al. solvents. 1997. A cyanobacterium was genetically engineered to produce very small amounts of ethanol some years ago (Deng and Coleman.. Gfeller and Gibbs. The more straightforward of these two is conversion by yeast fermentation of carbohydrate storage products. 1999).. Matsumoto et al. with only about 1 % of the biomass converted to ethanol. 1996. Miura et al. 1984. A ‘selffermentation’ of carbohydrate storage products by endogenous algal enzymes induced in the absence of oxygen has been reported for Chlamydomonas. glycogen. 2003.4 Hydrogen Production Photobiological hydrogen production has been investigated since the early 1970s as a potential source of this clean fuel (Prince and Kheshgi. However. with a physical pretreatment of the algal biomass required to make their intracellular carbohydrates available to enzymatic saccharification into simpler sugars (Hirano et al.Biofuels from Microalgae and Seaweeds 169 reducing ammonia levels through nitrogen sequestration into additional bacterial biomass (Samson and LeDuy.. the cost of biogas production by anaerobic digestion is relatively low. At present.. 2000. Another alternative would be to develop bacterial cultures that are more resistant to ammonia inhibition. Nath and Das. 2007). Such yeast-mediated fermentation of microalgal biomass has been demonstrated experimentally by several investigators. 1998. Benemann and Pedroni. This process is carried out either by anaerobic bacteria or by the microalgal cell itself via a selffermentation process as described above (Gfeller and Gibbs. 8. or even glycerol accumulated at high salinities by Dunaliella. Hirano et al. 8. 1987). Dark fermentation involves the anaerobic conversion of reduced substrates from algae.. but the achieved yields and concentrations of ethanol have not been reported. Methane generation by anaerobic digestion can be considered to be the default energy conversion process for microalgal biomass. 1983b. (ii) light-driven fermentations (also called photo-fermentations). such as starch. Yen and Brune. and mixed acids. such as starch in green algae. 1996. dark anaerobic hydrogen fermentations are limited to . 1996). and correspondingly low ethanol titers. the splitting of water into hydrogen and oxygen (Benemann. Several private companies are now reported to be developing ethanol fermentations by microalgae. Shirai et al. 1982. Ohta et al. Hirayama et al. or glycerol into hydrogen. yeast-based processes are economically unattractive because ethanol yields are quite low. Hon-Nami.

1981. 2002). 1984.1974). Under nutrient limitation.1973. Most current research is focusing on direct biophotolysis. but the overall hydrogen yields and light energy conversion efficiencies were far too low for economic viability (Akano et al.5 Oil Production Many microalgae. 2000). 1998. Here. 1997. but metabolic energy supply via photosynthesis is not (Roessler. in which water is split into hydrogen and oxygen without intermediate carbon fixation (Hallenbeck and Benemann. Sheehan et al. rather low yields due to thermodynamic constraints. photosynthetic oxygen evolution and carbon fixation into storage carbohydrates (starch or glycogen) takes place. These lipids can then be extracted from the biomass and converted into biodiesel or green diesel as substitutes for petroleum-derived transportation fuels.BenemannandWeare.170 Diesel from Biomass .. can accumulate significant quantities neutral lipids primarily as triacylglycerols (TAGs. Shifrin and Chisholm.2. with oxygen as a byproduct. is a mostly theoretical concept that remains to be demonstrated experimentally. Hydrogen can also be produced by microalgae via direct or indirect biophotolysis. Indirect biophotolysis. 1990).. oxygen and hydrogen production take place at different times or in separate stages. Suen et al.. 2002). 1997b. 2007). In indirect biophotolysis. . Piorreck et al. with only about 25 % of the energy in the starch being converted into hydrogen (Hallenbeck and Benemann. vegetable oils) (Sheehan et al.. 1989. In the first stage. with these then being used in a second stage for light-driven or dark fermenative hydrogen production process. however. Even if oxygen inhibition were to be overcome. with intermediate fixation and release of CO2 which is subsequently recycled (Hallenbeck and Benemann. 1998).. Ike et al.. this reaction is limited by the strong inhibition of the hydrogenase enzyme by the oxygen.. The primary challenge in developing an economically viable microalgal oil production process is to simultaneously achieve both high cellular oil contents and high oil productivities. no mechanism has yet been demonstrated that could be developed as a practical process for hydrogen production using microalgae (Benemann and Pedroni. Lipid biosynthesis is typically triggered under conditions when cellular growth is limited. Toconclude. such as by a nutrient deficiency.. Roessler. Nutrient deficiencies used to induce lipid accumulation include nitrogen deficiency for green algae and silicon deficiency for diatoms (Harwood and Jones.after35yearsofresearch(Benemannetal. the concept is to use microalgae to catalyze the conversion of solar energy and water into hydrogen fuel. 8. However. 2001).. These high-energy storage products can be present in amounts up to 30 % of the cell’s dry weight. 1977). direct biophotolysis would still suffer from the practical problems of generating explosive hydrogen–oxygen mixtures and the requirement for expensive photobioreactors to contain the reactions. 1997a. McGinnis et al. in particular green algae and diatoms. Takagi et al. 1987. 1990.. 1996. 2002). 1999. Organic acids formed during dark fermentations can be converted into hydrogen using nitrogen-fixing photosynthetic bacteria in a process called ‘photofermentation’ (Benemann. . Dark fermentation of microalgal biomass to produce organic acids followed by photofermentation for hydrogen production has been successfully demonstrated by several groups. Kawaguchi et al.

These concluded that a very favorable EOR approaching 10 is theoretically achievable (Benemann et al. 1996).5 GJ . and the supply water. and the greenhouse gas balances are clearly unfavorable (Farrell et al. This green microalga produces up to or more than 50 % of its dry weight as pure hydrocarbons. Wang. typically long tubes. 180 and 670 GJ haÀ1 yrÀ1. harvesting. Goldman and Ryther (1977) argued that energy inputs into large-scale microalgal cultivation systems for methane production would outweigh the energy gains – that is. Microalgae can also be mass cultured in closed photobioreactors. These authors projected that the algal biomass production is somewhat greater for the photobioreactors (58 mt haÀ1 yrÀ1 for tubular. typically between C20 and C40. Benemann and Oswald. but it is a clear target for further research. steel. the case of Botrycoccus braunii deserves special notice. 2006.6 Energy and Greenhouse Gas Input:Output Ratios For any biofuel to contribute to greenhouse gas mitigation.Biofuels from Microalgae and Seaweeds 171 the fraction of cellular lipids often increases but the growth and photosynthetic rates decline. for which the EOR is marginally above 1 (if that). 2006.. 2005. the energy for the biomass processing and biofuel production plant.. and the fuel conversion processes exhibiting variation from one plant to another. the mixing of the cultures. the energy inputs for crop cultivation. Existing analyses are based on conceptual engineering designs which remain to be validated. and not just the oil content. 1982. the overall result is that there is no increase nor decrease in oil productivity. The calculations involved in such analyses are difficult and so are often contentious. nitrogen and other fertilizers and other chemicals. This issue of energy inputs versus outputs in microalgal biomass open pond production was addressed by later studies based on many favorable assumptions. concrete. Recently. The key favorable assumptions were that it would be possible to produce algal biomass at productivities approaching 100 mt haÀ1 yrÀ1 with 40 % oil content in large. The major energy inputs to microalgal biomass production include the pumping of CO2 to and transfer into the algal culture. Pimentel and Patzek. these net energy and greenhouse gas balance issues are even more uncertain.e. 82 mt haÀ1 yrÀ1 for flat-plate designs) than for the raceway ponds (52 mt haÀ1 yrÀ1).). an EOR less than 1. which are site-specific. further studies are required to maximize lipid productivities.. etc. Oswald and Benemann (1977) rebutted this analysis by arguing that the EOR could be positive by recovering energy-rich fertilizers from the anaerobic digester and maintaining mixing velocities at 30 cm sÀ1 or less to minimize energy consumption. plastic bags or similar devices. with issues such as how to calculate and amortize embodied energy in the conversion facilities (e. it must substitute for fossil fuels and exhibit a net positive energy and greenhouse gas balance. They also pointed out that the energy required for mixing is much greater in the tubular and flat-plate photobioreactors (i. paddle-wheel mixed-raceway ponds. Finally. Rodolfi et al. 2005). Hence. with the energy outputs of algae grown in such systems. unlined. In the case of microalgal or seaweed biofuel production systems. respectively) versus only 6. and processing. as there are presently no large-scale or even pilot-scale operating systems. 8.g. (2007) compared the energy embodied in materials used to construct photobioreactors and consumed for mixing.2.. Hill et al. the harvesting of the biomass. the very low growth rate of this alga makes it impossible to mass culture. This has been an issue with corn ethanol. Unfortunately.

Somewhat over half of the current commercial production is of Spirulina. but this would likely not significantly affect the overall economics. and this has a value of US$ 13 GJÀ1. and that this could defray the entire production costs. Similarly. as is Chlorella in one plant in Germany. and Haematococcus pluvialis. even from this preliminary analysis it was clear that highly engineered photobioreactors would have a negative energy balance and. and has a global annual output of about 10 000 dry tons.172 Diesel from Biomass haÀ1 yrÀ1 required to operate the raceway pond. In Asia. as well as some producers of the other algae. Thus. a negative greenhouse gas account. and that TAGs have a value of US$ 1000 mtÀ1 (equivalent to about US$ 150 per barrel of oil). used to produce astaxanthin. 1900 versus 1200 GJ haÀ1 yrÀ1). It could be argued that. Chlorella vulgaris. in order of output. while Australian Dunaliella is produced in very large (100 ha) open unmixed ponds. Assuming further that the residual biomass after oil extraction is digested to methane. These production systems are small-scale models for the envisioned large-scale algal biofuels industry. they are at present the only plausible system for the production of biofuels by microalgae.5 GJ tÀ1 biomass (ca. by implication. Dunaliella salina. equivalent to a zero net energy output. while the highly engineered flat-bed photobioreactor has a greater gross biomass energy output than the simpler outdoor raceway pond (i.2. the EOR for the ponds is 10 while that for the photobioreactors is 1.. both capital and operating. cultivated in circular mixed open ponds. yielding an additional 7. the amortized energy content of the materials used when constructing the flat-bed photobioreactor was estimated at 1200 GJ haÀ1 yrÀ1. a rich source of beta-carotene. . with the remainder. but with some Chlorella.7 Microalgal Biomass Production Systems and Costs As there is not yet any experience with even pilot plants for microalgal biofuels production. with the raceway paddle-wheel mixed-pond design dominant in the industry and used by all Spirulina producers. another highvalue product. with perhaps a dozen companies producing several hundred tons each of various types of microalgae. The current microalgae industry produces algal biomass mainly in open ponds for human nutritional products. These four microalgae are produced in a variety of different production systems. About half of the world production of microalgae is in China. The residue following oil extraction may have additional value as animal feed or fertilizer. with both greenhouse gas abatement and renewable energy credits associated with a market for carbon. much of the Chlorella production is. While other factors such as the fertilizer and processing energy inputs were not considered. Assuming that one-third of the algal biomass consists of TAGs. 8. while it was only about 115 GJ haÀ1 yrÀ1 for the raceway pond. Given that open raceway ponds are also much more cost-competitive than closed photobioreactors. then the total value of the algal biomass could reach US$ 400 mtÀ1. 60 % of its energy content). the value of algal biomass used for biofuels could increase to US$ 500 mtÀ1 or more. that 90 % of these are recoverable. then the microalgal biomass has a value of US$ 300 mtÀ1. Haematococcus is produced in several countries in closed photobioreactors. consisting mainly of Spirulina. the economics of such a process must be based on current commercial experience with relatively small-scale production systems and conceptual engineering and cost analysis.e. however.

although promoted by many research groups and even private ventures in this field (Chisti. for high TAGs containing high value (> US$ 100 lÀ1) omega-3 fatty acids. also in the US. The detailed techno-economic studies quoted above. Benemann and Oswald. but it remains to be seen if the yields of oil can justify their sugar–oil conversion process. productivities in the range of 100 mt haÀ1 yrÀ1 will be necessary to achieve the above-stated cost goal of US$ 500 tÀ1 of high-oil algal biomass.5 ha in size. For example. Benemann). with plant gate production in the mainland US estimated at US$ 5000 per dry ton for a spray-dried product (not including selling. the tubular photobioreactor systems operating in Germany and Israel. The highest production costs are likely for Haematococcus pluvialis. but these are either not harvested or. but in this case the closed systems result in a twofold higher content of astaxanthin. The information presented here is based on industry contacts and personal knowledge by one of the authors (J. are disposed of (chemical flocculants inhibit methane production). The lowest production costs are attained for Spirulina. This is also the general design used in published techno-economic analyses for biofuels production. This annual output. at almost tenfold that of Spirulina. valued at US$ 50 000 haÀ1. 1996). each approximately 1 ha in size. raceway pond design. The production of TAGs for biodiesel and green diesel by algal fermentations has recently been promoted by Solazyme. Typically.and macroalgae from the wild for biofuels production. could justify a capital investment of between US$ 100 000 and US$ 200 000 haÀ1. but still require relatively long-term research in microalgal biology and production engineering. with little reliable published information.. there are no published studies or analyses. but these likely add up to not much more than a few hundred tons of biomass. such schemes are impractical and not further considered. most notably by Martek Corporation in the US. 1987. due to lower productivity and greater difficulty of cultivation. 2007). most importantly. with CO2 supplied via diffusers. cannot be considered for large-scale microalgae biofuels production. as used by most commercial microalgae producers.Biofuels from Microalgae and Seaweeds 173 Details on large commercial production systems are sparse. Hundreds of smaller algae production systems operate around the world for supplying the aquaculture industry. For microalgae biofuels production. and there are proposals for harvesting both micro. It is also worth noting that several other . microalgae are also grown by fermentation in the dark on sugars for the production of Chlorella in some plants in the Far East and.2 and 0. However. this makes the biomass more valuable and also significantly reduces the extraction costs. when recovered from the pond effluents by chemical flocculation. These analyses suggest that.or concretelined and are between 0. these are rough estimates based on knowledge of the industry. closed photobioreactors. overhead or capital costs). only the open. 1982. paddle wheel mixed. these ponds are plastic. except that the ponds would be at least tenfold larger and lined with less-expensive clay (Benemann et al. were reported to have cost many millions of dollars. and an operating cost of between US$ 100 and US$ 200 tÀ1. Algae are also grown in wastewater treatment oxidation ponds. Weissman and Goebel. even with the lowest possible projected capital and operating costs. The reason is the high cost of such systems. Microalgae are removed from drinking water reservoirs by means of screens. Finally. Again. let alone the cost of the fermentation equipment and operations. has the required combination of potentially high productivity and low costs. This alga is also produced commercially in closed photobioreactors. suggest that such cost goals are achievable. Finally. at perhaps tenfold the cost of open ponds.

1988). It might be argued that current commercial experience has little relevance to future biofuels production. temperature control. personal information). and that biotechnology and improved engineering will overcome current limitations. pharmaceutical. per unit area. despite some claims to the contrary (Chisti. These Table 8. and Limitations Introduction The global annual harvest of wild and cultivated macroalgae. biological/microbiological) . food additive. Cleaning. with flat-plate and dome reactors having even smaller maximum unit sizes of a few square meters. toothpaste) Other uses of seaweeds . Processes. However. a fundamental analysis demonstrates that. Benemann. tubular closed photobioreactors systems cannot readily be scaled up beyond about 100–200 m2 (Weissman et al. of such photobioreactors into one large system. and other management chores are multiplied manyfold. but these failed for operational and economic reasons (J.3 8.174 Diesel from Biomass large systems have been operated in the past. or seaweeds. medical) . with a value of about US$ 6 billion in 2003 (McHugh.0 billion .1 Biofuels from Seaweeds: Products. 2007). 1 million dry tons). Agar (food ingredient. manyfold more expensive to build and operate than open ponds. such as in Spain for Dunaliella production and in Argentina for Spirulina production.) Algal hydrocolloids .. due to O2 accumulation and gasexchange limitations. and even hundreds or thousands. Macroalgal biofuels Total Value (US$) 5 billion 132 million 213 million 240 million 5 million 5 million Negligible 5. Alginate (textile printing. 8. etc. Indeed.2 Global value of seaweed products per annum (from McHugh. with perhaps 50 % growth since then. 2003) Product Human food (Nori. The extraction and use of chemicals for polysaccharide-based hydrocolloids represented an annual portfolio of products valued at US$ 585 million (Table 8. was 7. wakame. They are also unaffordable for biofuels production. aonori. each with individual controls. pharmaceutical. but that does little to reduce the economic penalty of building and operating such large numbers of reactors.5–8 million metric tons wet weight (ca. 2003). many new commercial entrants in this field – most of which are focused on oil production – are developing various photobioreactors designs. kombu. most of this is consumed as human food.5–6. In conclusion.3. and technical oversight. Animal feed . pet food.2). it is possible to combine tens. monitoring equipment. Of course. dilution pumps. closed photobioreactors are. even assuming that they had higher productivities and were less subject to contamination than open ponds. Fertilizers and conditioners . Carrageenan (food additive.

The potential for this marine biomass as feedstock for conversion to methane is reported to be greater than 100 EJ yrÀ1. An early concept for open-ocean seaweed farming for energy proposed in the early 1970s was aimed at deriving value from the floating seaweed Sargassum (J. The concept of this latter project was very ambitious including. in addition to arrays for growing seaweed. By comparison. personal communication). seaweeds do not contain extractable oils. The cost of producing methane from seaweed biomass was calculated to be equivalent to that of methane produced from the conversion of terrestrial feedstocks such as sorghum or poplar. According to their analysis. exceeding by over threefold the total that could be generated by the conversion of other sources of biomass (Chynoweth et al. Although. 2001).. 2002). 2001). Juvenile Sargassum would be released about 500 miles off the west coast of the US. This initial concept on oceanic Sargassum cultivation was the precursor of more detailed proposals for the open-ocean production of seaweeds for energy. wave-powered upwelling pumps. The history of growing algae in offshore energy farms includes major efforts undertaken in the US during the 1970s to early 1980s. the favorable projections of the energy conversion process noted above. a seaweed processing plant with living quarters for the crew. Korea and other parts of Asia with a history of economic uses of seaweeds. agar and carrageenans. unlike microalgae. such as the United Kingdom (House of Commons. seaweeds are attracting increasing attention as a potential energy source. which points to the very large scale at which such an operation would need to be run in order to make a significant impact. facilities for ships. For some coastal European nations. butanol. Although neither of the preceding concepts was implemented. even though the potential of seaweeds as feedstock for biofuels has been recognized for several decades (Bird and Benson. Waiting ships would harvest and transport the algae to onshore anaerobic digesters for conversion to methane. the economic contribution of seaweeds as a source material for producing renewable fuels is negligible. Any contemporary consideration of seaweeds for biofuels requires a re-examination of the activities that took place as part of this effort. 1982). which are used not only as gums and thickening agents in foods and feeds but also have industrial applications such as in textile printing (McHugh. 2001. Although less so than for microalgae. exceeding US$ 30 million (Bird and . The primary deterrent to the optimistic projections based on maximal productivities are the many uncertainties in the design of offshore farms and the production of an adequate supply of algal feedstock (Chynoweth et al. 2003). and advances in offshore technology are among the fundamental factors encouraging new ventures in this particular arena.. their high carbohydrate content suggests that they might be utilized as feedstock for ethanol. They would then be carried south by existing currents to the latitude of the US–Mexico border. seaweeds are viewed as a possible avenue to help meet CO2 reduction targets. There also is renewed interest in Japan. thus making seaweeds a competitive primary raw material for manufacturing biofuels (Chynoweth et al. growing to harvestable size during the interim. all of the US energy needs could be covered by biofuels derived from marine macroalgae grown on about 243 million hectares (one million square miles) of ocean. Nevertheless. and a helicopter platform. 2006). they served as the genesis of the Marine Biomass Program. or other fermentations. at about the latitude of the US–Canadian border. 1987). one of the largest single investments made by the US Department of Energy during the period 1979 to 1983. as described for the ‘Ocean Food and Energy Farm Project’ (Wilcox.Biofuels from Microalgae and Seaweeds 175 include alginates. the current economic and environmental considerations. Chynoweth. Benemann..

1987). 1987). This investment was meant to constitute an initial installment towards the construction of a 40 000 ha. 50–70 tons of ash-free dry weight per hectare per year (Hanisak. and this resulted in the loss not only of algae from the structures but also the structures themselves. such enclosed net systems are still hypothetical. significant advances were made in understanding the biological requirements and associated complexities of growing seaweed in an open-ocean environment using such platforms. possibly with recycled nutrients. These studies also suggested that productivities would require a relatively large energy input to reduce diffusion limitation by CO2 and other nutrients. Resolution of the following major technical issues is needed to achieve open-ocean farming of seaweeds: 1. These should prove useful as lessons for future efforts in open-ocean seaweed cultivation. which would be floated in the open ocean in natural upwelling zones where nutrients are freely available. The program had as its major focus several trials with large floating platforms to grow the giant kelp Macrocystis pyrifera. Many innovative systems are being proposed. US$ 2 billion open-ocean farm (Neushul. may not be critical if the system is affordable and robust. Nutrient supply and uptake: The concept of artificial nutrient upwelling is one that has yet to be demonstrated as technically feasible. Containment. it became clear from work with onshore systems that it should be possible to attain high productivities for the offshore farms. and the ability to engineer and manage such large farms in an open ocean environment remains to be demonstrated. For future endeavors in open-ocean seaweed cultivation. bringing the conversion of marine biomass to biofuels to fruition is a long-term project. 2. 1987. as practiced in Asia. and extreme conditions associated with storms (McHugh. If not supplied with upwelled nutrients the plants must be artificially fertilized. with an extension to large-scale farms in the open ocean still at the conceptual stage. in particular the use of nutrient-rich. with the program coming to an end in 1983. However.176 Diesel from Biomass Benson. Moreover. Productivity: The actual productivity. whether it is 10 or 100 mt haÀ1 yrÀ1. the structural design of the platforms and the procedures used for attaching the seaweeds could not withstand the dynamic open-ocean environment. An operational farm was never realized. additional engineering research is needed on the design of suitable structures that will allow the seaweeds to survive dynamic oceanic conditions such as currents. generally based on net enclosures. some as large as a square kilometer. upwelled seawater. 1987). with major issues of plume dispersion. Nevertheless. However. During this time. Hanisak and Samuel. and distribution: It is clear that no engineering design has thus far been demonstrated that can contain the plants and provide them with protection from storm and other damage. while funding for research and . protection. sinking. that is. Its focus was research into the provision of nutrients to the plants. could be advantageous. wave movements. Growing algae nearshore. 2003). the first of which was installed in September 1978 (North. and even CO2 releases. 1987). protection and distribution issues noted above. the present technology for seaweed cultivation is limited to near-shore systems. Thus. where currents provide significant mixing. The push for higher productivities likely reflects a lack of ability to address the fundamental engineering and economic issues associated with the containment. 3. The net would be capable of being lowered below the wave action zone during storms.

such kites.Biofuels from Microalgae and Seaweeds 177 development has been scaled down over the past two decades. including the production of electricity and liquid transportation fuels such as ethanol from seaweed. Yet recent activities. In the UK and Ireland.3. In order to boost the efficiency of the overall process. led by the Scottish Marine Association to investigate the economics and feasibility of culturing algae for production of third generation biofuels such as methane and ethanol (see www.’ is to produce 5 billion liters of bioethanol. with a length of 1.3 Seaweed to Ethanol Ethanol can also be produced from seaweed (Horn et al. Different designs using a soft facility structure comprised of ropes and nets are planned for these environments.8 kW generation demonstration facility. combined with improvements in seaweed farming for aquaculture and the potential for microalgae as a optimization is still needed to enable industrial implementation (Horn et al. The objective is to create an offshore farm on 4. . The best yield of ethanol attained to date using seaweed as the raw material is 0. Ocean currents would maintain the open triangular configuration of the seaweed attachment array. By using this process. approximately 20 kl methane per ton seaweed per day can yield up to 10 kW hÀ1 of electricity.. Farming in deep waters as planned would use ‘sea kites’. referred to as the ‘Ocean Sunrise Project. both of which are byproducts of alginate production. Extracts from Laminaria hyperborea can be fermented to ethanol by conversion of mannitol and laminaran. For this. Likewise. 8. horneri for producing bioethanol (Aizawa et al.47 million km2 in Japan’s exclusive economic zone to grow km would be moored to the sea bottom using modern deep-water mooring technology. the Sustainable Fuels from Marine Biomass project (BioMara)..2 Anaerobic Digestion of Seaweeds The production of methane from seaweed was studied in the US Marine Biomass program (Chynoweth.5 km and a width of 1. and has recently been demonstrated at the pilot scale in Japan. The envisioned system is to be located both in coastal zones at depths less than or equal to 500 long-term goal of this plan. through a collaboration between Tokyo Gas and the Japanese government agency New Energy and Industrial Technology Development Organization (NEDO) (http://web-japan. proof of concept of the anaerobic digestion of seaweeds for methane production achieved in early studies by Chenowyth and colleagues. the methane product can be blended with natural gas and subsequently converted into electricity. Nonetheless.3. Farming in coastal zones will use established methods currently used to farm Laminaria and Undaria for food products.html). Tokyo Gas demonstrated this concept by operating a 9. 2000). 2002). the seaweed Sargassum horneri could be a source of raw material for the manufacture of liquid biofuel feedstock using a bacterial consortium that has been identified as optimal for this bioconversion.. 8.43 g gÀ1 substrate in batch culture.biomara. have led to formation of a consortium. and offshore in partially oceanic areas but in waters between 500 and 3000 m deep. are now under investigation. seaweed washed up on shore was used. The mid. using the bacterium Zymobacter palmae and the yeast Pichia angophorae. 2007). 2000). some examples of these efforts are described below.

which resulted in blades of 1. the technology is transferable to production of seaweed as a biofuel feedstock.5 Seaweed Biotechnology Although seaweed biotechnology is at an early stage of scientific and technical development.5–2 m in length when placed in offshore waters (Buck and Buchholz. 2003).178 Diesel from Biomass Danish scientists are currently assessing the feasibility of conversion to ethanol of the green alga Ulva lactuca. 2005) In order to attach the plants to culture lines.3. Envisioned are large-scale farms that would grow seaweed in artificial coastal ponds. rather than lashing or sewing holdfasts to the lines. 2001). The annual production potential is projected as 80 000–100 000 t. 8. with a theoretical yield of 200–500 t wet biomass per hectare. 2007). as is the scale of farming operations needed to measurably impact the demand for fuels.Centre National de Squencage (France). The ring structure was found to be stable in offshore conditions and to support the seaweed growth. The average biomass grown on the ring is about 4 kg mÀ1 culture line.3. At present. that is. the stack effluents of which would be diverted to supply CO2. 1996. Ulva lactuca is reported to contain up to 60 % carbohydrate. which was based on lashing seaweed to support structures for continuous harvest and regeneration within the offshore farm. and would require ponds to be placed in the vicinity of power plants. 2004). Transformation systems have also e e ¸ been developed for several species of seaweeds. Advances in protoplast fusion protocols. 2004). which allow recombination between different algal genomes . The latter is believed to be achievable with intensive farming with added nitrogen fertilizer from animal wastes and CO2. it was found that this species could withstand current velocities of 1. the first genomesequencing efforts for seaweeds are under way for the red alga Porphyra purpurea at the Joint Genome Institute (US DOE). Jiang et al.. 2002. 1 cm).4 m. This process of seeding and deployment of culture lines differed from the technology developed within the framework of the Marine Biomass Program. conditions that are equivalent to storm conditions in the North Sea (Buck and Buchholz.52 m sÀ1 and waves of 6. 8. Huang et al.4 Offshore Seaweed Cultivation Technology Only a few of the designs for structures for offshore seaweed farming have moved to a development and testing phase. While the stated aim for developing the structure was for the food industry. 2003. For example. When tested for its resistance to hydrodynamic forces.. important advances are currently being made. and the brown alga Ecotocarpus siliculosisus at the Gnoscope . which grows abundantly along Danish shorelines (The Trade Council.. the economic feasibility of this scheme is unknown. One notable example is the offshore ring system developed by German scientists to produce Laminaria saccharina for food (Buck and Buchholz. a figure comparable to that for wheat or maize. The genus Laminaria is among the seaweeds that can be converted to methane in a costcompetitive manner as compared to terrestrial biomass and municipal solid waste (Chynoweth et al. thus making it a candidate for the production of bioethanol. prior to their placement on the offshore structure. the sporophytes can be cultured in the laboratory and allowed to settle on the lines and grow to a suitable length (ca.. making it possible to create genetically altered strains (Gan et al.

the number of studies on the formation and regeneration of calluses and protoplasts in seaweeds is rapidly growing. 1998). CO2. The technology for large-scale. while gaining increased recognition. The most significant challenge to using algae as an alternative renewable energy source is cultivating them at a scale that would make a significant impact on the global energy economy. 2003). There are. 100-fold higher than are required for biofuels production. Although seaweeds are already a significant economic resource (McHugh. Chynoweth et al. on land or the open ocean. However. has yet to be realized.. For example. water. and competing societal needs limit the extent to which near-shore environments can be used for large-scale seaweed cultivation. The production potential for both types of algal biomass is far from being realized..g. zoospores of Laminaria have been successfully used to seed ropes used for the offshore rings discussed above (Buck and Buchholz. Similarly. no simple. major uncertainties remain in all aspects of the design and operation of these biomass production systems and. Thus. or sufficient solutions to . Today. has not been sufficiently assessed. have the potential to engineer seed stock with characteristics favorable for growth in dynamic offshore environments. Providing nutrients to such farms. such technologies will need to utilize naturally upwelling nutrients for open-ocean seaweed production or waste nutrients for land-based microalgae systems. Thus. 8. The micropropagation of seaweeds using algae taken at early developmental stages in their life history is a well-established practice in the seaweed aquaculture industry.. The potential for growing algae as an energy feedstock compares favorably with other plant biofuel resources. environmental. with costs ranging from 10. the biomass to biofuels conversion processes themselves.Biofuels from Microalgae and Seaweeds 179 without altering existing genes (Cheney et al.. despite the potential availability of land. the commercial production of microalgae is a very minor activity. Coastlines are themselves a limited resource. microalgae production on land-based pond systems is severely limited by the need for a juxtaposition of suitable land. the technology is at present capable only of producing high-value products. either by pumping deep ocean waters or providing synthetic fertilizers would be prohibitively expensive and environmentally unacceptable. and social challenges that need to be addressed are formidable. 2003). climate and nutrient resources. Lastly. open-ocean seaweed farming has yet to be developed and demonstrated as being technologically and economically feasible. certain. coastal and oceanic resources suitable for their production. however. Neither open-ocean farming nor on-shore ponds for algal production using power plant flue gases has yet been demonstrated at even the pilot scale. and the use of such techniques for seaweed breeding is likely to contribute to selecting desired characteristics for bioconversion and for open-ocean seaweed culture. according to several economic projections (e. Sheehan et al. the ecological impact of deployment of such installations. to a lesser degree. the technical.4 Perspective The potential of microalgae and seaweeds as feedstock for conversion to biofuels. Scientific and technological challenges remain to be addressed before these sources can be considered economically viable for the production of renewable fuel. 2004). 2001. For both seaweed production off-shore and microalgae production on shore.

Vol. Benemann J. Hydrogen production by microalgae. Kamen. Washington. Hydrogen biotechnology: Progress and prospects. Applied Biochemistry and Biotechnology 57/58: 677–688. Miyasaka.pdf. 2000.R. Canada. J. T. and J. Strain manipulation and improvement in the edible seaweed Porphyra. Buck B. DC. Elsevier Science Ltd.C. in Living Systems as Energy Converters. 1977. 1996. Northeastern University. In this chapter we have briefly highlighted the challenges that must be addressed to make both micro.H. Miura.531. In U. Roberts K. and T. Biofixation of Fossil CO2 by Microalgae for Greenhouse Gas Abatement. Washington.osti.S.M. and N. and greenhouse gas abatement. T.jsp?query_ id¼0&page¼0&osti_ id¼6374113. 1982.O. H.H. Vancouver. R.). Pedroni. Amsterdam. Benemann J. 6.and macroalgae a source for a viable path to renewable biofuels production.H. N. P.M. Weare. U. The Netherlands. Goebel. 2003. Hydrogen evolution by nitrogen-fixing Anabaena cylindrica cultures.. .R. 2007.. 2004. and I. Amsterdam.P. 1996. Benemann J. Nature Biotechnology 14 (September): 1101–1103.S. 1974. Y.M. Akano T. Seaweed Cultivation for Renewable Resources. A. N. Technical Report DOE/ER/30014-T1. pp. Response of offshore cultivated Laminaria saccharina to hydrodynamic forcing in the North Sea. K. Asaoka. Benemann J.T. 1996. Yagi. Systems and Economic Analysis of Microalgae Ponds for Conversion of CO2 to Biomass. 396 pp. Journal of Applied Phycology 12: 291–300. Augenstein. Balancing these points are the potential benefits that can flow from developing these technologies. U. The offshore-ring: A new system design for the open ocean aquaculture of macroalgae. Available at: http:// govdocs. 1973. and W.. a. Department of Energy. Maeda. Atsumi. Hydrogen evolution by a chloroplast-ferredoxin-hydrogenase system. Microalgae As a Source of Liquid Fuels. H. 2007 Seaweed bioethanol production in Japan – The Ocean Sunrise Project. Kaplan. J.. Patent and Trademark Office. Cheney. Bird K. Journal of Applied Phycology 16: 355–368. Benemann J. Science 184: 1917–1918. K. the future use of which will be limited both by declining supplies and increasing environmental costs.R. Department of Energy. Environment International 24(8): 889–897.. Aquaculture 250: 674–691.J. D.. and P. Benemann J. Weissman. Available at: http://www.L. together they could substitute for much of the current demand for fossil fuels. Benemann J. References Aizawa M.D. 1987. Shioji. Buvet et al. Oswald. Fukatsu. Buchholz. R.. waste treatment..C. Berenson.R. Y.P.R.M.biblio. DC. neither will any other single biofuel approach provide a solution to our energy problems. U.J. Oswald. Treccani Encylopedia of Hydrocarbons Volume III: pp. Hydrogen and methane production through microbial photosynthesis. M. Thermochemical treatment for algal fermentation. and 837–861. 2005. Hydrogen production by photosynthetic microorganisms. and W. and C. and Watson K. Mizoguchi. Buchholz.A.aquake. (eds). S.646 (ed. U.M. and C. 285–298. not only for production of biofuels but also for other higher-value commodities.A. Yet. from animal feeds and human foods to valuable fertilizers. Proceedings of the National Academy of Sciences (Washington) 70: 2317–2320.180 Diesel from Biomass the impending energy supply problems. Sakou. Elsevier/North-Holland Biomedical Press. 1998. and J.R. Hamasaki. Matsumoto. K. Chen P. Ikuta. Buck B. and all potential resources should be investigated to at least the stage at which their feasibility and potential is fully understood..S. Although biofuels production from algae is not likely to be a major energy resource. Benson. Benemann in Oceans 2007.R. chemicals. Office.

Gan S. R. Methane fermentation of microalgae. Mathieson.J. 1984. Gotaas. R. Nelson. Hill J.I.T. Ltd.P. HC 965-I.E. S.M.Z. International Journal of Hydrogen Energy 27: 1185–1193. Cambridge University Press. A. and Y. R. USA. Hon-Nami K. Growth-rates in culture of several species of Sargassum from Florida.L. United Kingdom. pp.. Transient expression of lacZ in particle bombarded Gracilaria changii (Gracilariales. and S. pp. and W. I. 1988.. A report prepared for Tokyo Gas Company. 2006.R.. Applied Biochemistry and Biotechnology 131(1–3): 808–828. 1997. 191–218. Harwood J. 1987. 2000. Eighth Report of Session 2005-06. Turner. and A.M. 1987. T. R. Review of Biomethane from Marine Biomass.H. Kyoto.B. Ueda.P. Othman. Transient expression of the GUS reporter gene in the protoplasts and partially digested cells of Ulva lactuca L (Chlorophyta). September 7–11. 1998.. Goebel. Hirano A. E. Samuel. S.J. Applied and Environmental Microbiology 65: 523–528. Plevin. K. S. Science 311: 506–508. Chynoweth D. 1999. C. Phang. Hughes (eds). Elsevier. J. Food and Rural Affairs Committee.J. Gunaseelan V.P. 1997.K.A. A. Applied Science Publishers Ltd. Tiburzi. Golueke C. in Algae and Human Affairs. Gibbs. Oswald. Ethanol production from carbon dioxide by fermentative microalgae.. Climate change: the role of bioenergy. Tilman. S. and D.. Jones.G. A unique feature of hydrogen recovery in endogenous starch-to-alcohol fermentation of the marine microalga. Minocha.L. 1957. 2007.Biofuels from Microalgae and Seaweeds 181 Chisti Y. D. and T. Hallenbeck P. Hon-Nami... J. Advances in Botanical Research 16: 1–53. and J. 2006. Biomass and Bioenergy 13(1/2): 83–114. Hirayama S.I. Bird and P. Ethanol can contribute to energy and environmental goals. Benson (eds). in Biological Solar Energy Conversion. Mitsui.M. Rhodophyta). Farrell A. and K. Owens.R. Proceedings of the National Academy of Sciences. in Anaerobic Digestion. Hirayama. A. House of Commons. Tamura (eds).A. Plant Physiology 75: 212–218.E.C. 1997. Harlin M. Ogushi. O’Hare. Huang X.H. M.J.D.. D. Japan.N.. Paper read at the Fourth International Conference on Carbon Dioxide Utilization. Lembi and J. Y. Kunito.Y.E. Y.T. Renewable Energy 22: 1–8. Energy 22(2/3): 137–142. Goldman J. and M. Fermentative metabolism of Chlamydomonas reinhardtii . Botanica Marina 39: 467–474. 1996. Anaerobic digestion of biomass for methane production: a review. B. Benemann. and H. D. Mass production of algae: bioengineering aspects. Biodiesel from microalgae. 1979. 3–27. Eisenberg D. Jones. Amsterdam. Journal of Industrial Microbiology & Biotechnology 25: 249–254. Yu.P. Oswald. and S. and energetic costs and benefits of biodiesel and ethanol biofuels.. Environment.Y. A. in Seaweed Cultivation for Renewable Resources. 1977. Biotechnology Advances 25: 294–306. Coleman.. Hinson. Deng M. and J. 2002. Tiffany. Legrand. 2006. The algae: an overview. 1996. Anaerobic digestion of algae. and R.. Hanisak M. Miyachi. Waaland (eds). 2003.T. and M.S. 2001.R. Hirano.C.M.M. J. Journal of Applied Phycology 15: 351–353. Academic Press. New York. .A. Environmental. Benemann. Ethanol synthesis by genetic engineering in Cyanobacteria. Analysis of fermentative products from starch in the dark and light. economic. B. and D. 1989. Darley. Chlamydomonas perigranulata. W. W. Hanisak M..-D.. Lipid metabolism in algae. A.C. Ethanol production from seaweed extract. Cultivation of Gracilaria and other macroalgae in Florida for energy consumption.M. Kammen..C. Pietro and S. Ueda. CO2 fixation and ethanol production with microalgal photosynthesis and intracellular anaerobic fermentation. Polasky. Chynoweth D. R. and S. Horn S.C. London. Ogushi.. Weber. USA 103(30): 11206–11210. Gfeller R. Aasen. Ostgaard. Wheatley and D. Applied and Environmental Microbiology 5(1): 47–55. Samejima. and J. Stafford. Ryther.D. Biological hydrogen production: Fundamentals and limiting processes. Qin.D. Renewable methane from anaerobic digestion of biomass. 2002. Hydrobiologia 151: 399–404. K.

Jassby A. Wu. 1997a. Mitigation and Adaptation Strategies for Global Change 11: 539–577. Kadam K. A. Italy. D. Biotechnology and Bioengineering 24: 1555–1563.. Sommerfeld. H. A. Hirata. N. C. 1995. Ikuta. and D. 1999. N. Wind. and T. and K. Fast pyrolysis of microalgae to produce renewable fuels. 2004. Y. Tolbert. Miyamoto. Miao X. Hashimoto.H. Yang. K. McHugh D. H. Plant Cell Reports 21: 1211–1216. Energy 27: 905–922. Nakano. 1982. Tsukamoto. N. M. Matsunaga. K. Journal of Bioscience and Bioengineering 88(1): 72–77.K. Lynd. British Bioenergy News 5: 16–17. and K. Spirulina: a model for microalgae as human food.R. High-value renewable energy from prairie grasses Environmental Science and Technology. Endo. Miao X.B. McLaughlin S..K. Sawayama. N. Matsumoto H. Miyamoto.. Y.. and D. 2004.A. Hirata. Ike... Wolf. K. C.E.. and S.182 Diesel from Biomass Huesemann M.D. Miura Y. K. Yagi. Miyamoto. Garten. Murakawa. Wu. Journal of Fermentation and Bioengineering 84(5): 428–433. Rome. Environmental implications of power generation via coal-microalgae cofiring. Cambridge University Press. Hydrogen production from algal biomass by a mixed culture of Rhodobium marinum A-501 and Lactobacillus amylovorus. Journal of Analytical and Applied Pyrolysis 71: 855–863.J. 1997b. Journal of Fermentation Technology 59: 441–446. Shoga. Miyamoto. and T. 2002. 1999. 2006. 1981. Kishimoto. Fuel 12: 1735–1738. Ike. and C. Chinese Scientific Bulletin 47: 1438–1440. 2006. and K. Lembi and J. Waaland (eds). Minowa T. and K. Toda. 2003. Yokoyama. T. K. 1988. Ike. and T. Applied Biochemistry and Biotechnology 51/52: 681–692. . Applied Biochemistry and Biotechnology 105–108: 247–254. N. Jiang P. Photoproduction of hydrogen from raw starch using a halophilic bacterial community. Suzuki.T. Miura Y. Expression of hepatitis B surface antigen gene (HBsAg) in Laminaria japonica (Laminariales.. 2004.A. Hirata. A. and C. Hydrogen photoproduction from CO2fixing microalgal biomass: application of halotolerant photosynthetic bacteria.. K.. McGinnis K. Hirata. Kawaguchi. High yield bio-oil production from fast pyrolysis by metabolic controlling of Chlorella protothecoides. Chamydomonas reinhardtii. Expression of the lacZ reporter gene in sporophytes of the seaweed Laminaria japonica (Phaeophyceae) by gametophyte-targeted transformation..L. Journal of Fermentation and Bioengineering 84(6): 606–609. Shioji.. 2003. Hydrogen photoproduction from CO2-fixing microalgal biomass: application of lactic acid fermentation by Lactobacillus amylovorus.-Y. Requirement of oxygen for dark hydrogen evolution by a green alga. Toda.G. Fuel 78: 1213–1215. and Q. Oil production from algal cells of Dunaliella tertiolecta by direct thermochemical liquefaction. pp. Hamasaki. Tseng. Phaeophyta). Food and Agricultural Organization of the United Nations. Sanderson. wave. M. A Guide to the Seaweed Industry. A. in Algae and Human Affairs. Miyamoto. Journal of Biotechnology 110: 85–93. Chlamydomonas reinhardtii. Fukuda.R. 36(10): 2122–2129. Kelly M. Miyamoto.R. 2002. M.A.. Can advances in science and technology prevent global warming? A critical review of limitations and challenges. H. K. Tsuji.M. and K. Ohata. Hydrogen production by a green alga. Y. Yokouchi. Sato.. Matsumoto M. N. Tseng. 2002. Okakura. Yagi. S. Q. Improvement of fermentative hydrogen production: various approaches. Jiang P. Journal of Applied Phycology 9: 19–24. 2001. Characterization of the growth and lipid content of the diatom Chaetoceros muelleri. S. 149–179. Qin. and M. Das. De La Torre Ugarte. Qin. Journal of Bioscience and Bioengineering 91(3): 277–282. Applied Microbiology and Biotechnology 65: 520–529. 1997. S. M. Nath K. and weed. in an alternating light/dark cycle.. 2003. and C.. and K. FAO Fisheries Technical Paper 441. Carbon dioxide fixation by microalgae photosynthesis using actual flue gas discharged from a boiler. Dempster. A novel microalgal system for energy production with nitrogen cycling. 1995. V. L.. Minowa T. Kawaguchi H. T. Saccharification of marine microalgae using marine bacteria for ethanol production.

LeDuy. Stokes. 379–396. 2001b.. Zilio-Grandi. 2007. Tu. Oswald W. and outdoor cultivation in pilot photobioreactors. K. W. Anaerobic digestion of nitrophilic algal biomass from the Venice Lagoon. Pohl. R. Q. and wood. 2004. Baasch. Golden. A Look Back at the U. Biotechnology Letters 5(10): 677–682. K. 2000.R. Sulser. Biotechnology Letters 5(10): 671–676. Pimentel D. and P. 2005. Mitsui. chlorophylls. 1986.osti.. in Biological Solar Energy Conversion. Benson (eds). Benson (eds). Yokoyama. A. Germany.W. Pyrolytic characteristics of heterotrophic Chlorella protothecoides for renewable bio-fuel production. Rismondo. 1984. 1990. 1987.S. Turhollow. Kheshgi. Improved performance of anaerobic digestion of Spirulina maxima algal biomass by addition of carbon-rich wastes. W. Sawayama S.L. 1987. 1–37. and J. Rigoni-Stern S. pp. A. Biomass as Feedstock for a Bioenergy and Bioproducts Industry: The Technical Feasibility of a Billion-Ton Annual Supply.. Q.H. 39–67. S. December 2006. Elsevier. Tu.Biofuels from Microalgae and Seaweeds 183 Neushul P. Energy from marine biomass: the historical record. total protein. A.-Y. 1998. and Shifrin N.G.T. 2020 Focus 14 Report. Hydrogen evolution as a consumption mode of reducing equivalents in green alga fermentation. Amsterdam. pp. 1981. B. Influence of mechanical and thermochemical pretreatments on anaerobic digestion of Spirulina maxima algal biomass. and S. Journal of Phycology 26: 393–399. T. Colorado. Oak Ridge National Laboratory. S. Phytoplankton lipids: interspecific differences and effects of nitrate. Biomass production. ORNL/TM-2005/66. Bassi. T. Bird and P. Bioresource Technology 80: 1–7.C. Wu. Prince R. Perlack R. Pyrolytic characteristics of microalgae as renewable energy source determined by thermogravimetric analysis. Tredici. Natural Resources Research 14(1): 65–76. 1987. Journal of Phycology 17: 374–384. R. Available at: www. 1990. San Pietro.J. Effects of temperature and holding time on production of renewable fuels from pyrolysis of Chlorella protothecoides. Journal of Applied Phycology 13: 5–12. Minowa.. Oak Ridge. Zhao. Oceanic farming of Macrocystis. K.T. Phytochemistry 23: 207–216. 1999. Miura. R. Tennessee.-H.A.K. and A. in Seaweed Cultivation for Renewable Resources K. N.W. Ohta S.L. 2001a. New York. in Bioenergy and Agriculture: Promises and Challenges. LeDuy. Elsevier. and S. Samson R.C. Tamura (eds).. 1977.. F. the problems and non-problems. 2005.S. Biondi. Plant Physiology 83: 1022–1026. Vigato. Wu.. Miyachi. Q. Valmonte-Santos. A critical analysis of bioconversion with microalgae. Brief #3. Benemann. and A. Patzek. Peng. North W. Journal of Applied Phycology 12: 147–152. Environmental control of glycerolipid metabolism in microalgae: commercial implications and future research directions. and light-dark cycles.R. T.C. Biotechnology and Bioengineering 28: 1014–11023. Bonini. and Y. National Renewable Energy Laboratory. Ethanol production using corn. Padovani.D. Wu. Biofuels and the global food balance. Academic Press. Tu. Biodiesel production using soybean and sunflower. Sheehan J.S. Berlin. LeDuy. The photobiological production of hydrogen: Potential efficiency and effectiveness as a renewable fuel.. 1983a.F. Detailed study of anaerobic digestion of Spirulina maxima algal biomass. and N..W. Pulz O. Zittelli. Wright. Roessler. Critical Reviews in Microbiology 31: 19–31. Amsterdam. International Food and Policy Research Institute.. Applied Microbiology and Biotechnology 65: 635–648. 2005. Hazell and R. 1983b. silicate. induction of lipid synthesis. Bird and P. Msangi.. lipids and fatty acids of freshwater green and blue-green algae under different nitrogen regimes.. and D. and P. Benemann. Pachauri (eds). and P. Biomass and Bioenergy 17: 33–39. June 11–14.. Peng. Graham. and A. Gross.. G. Rosegrant M. L. 2006. Possibility of renewable energy production and CO2 mitigation by thermochemical liquefaction of microalgae. and P. Piorreck M. G. and H. and T. Samson R. pp. in Seaweed Cultivation for Renewable Resources. and R. N. Peng W. Department of Energy’s Aquatic Species Program – Biodiesel from Algae. Rodolfi L. Dunahay. G. J. and P.. 2007. Roessler P.. Lipid production from marine microalgae: Strain selection. Samson.H. P. Presented at the 7th European Workshop on Biotechnology of Microalgae.. Erbach. Valuable products from biotechnology of microalgae. Biomass 23(3): 179–199. . P.J. Chisholm. switchgrass. Miyamoto. and W. and S. L. Szpyrkowicz.

A renewable energy source – hydrocarbon gases resulting from pyrolysis of the marine nanoplanktonic alga Emiliania huxleyi. Yamaberi.. Weissman J.-W.. Mizuki. The Trade Council. 2005. Suen Y. and J. Wanatabe. Solar Energy Fixation and Conservation with Algal-Bacterial Systems. Ministry of Foreign Affairs Denmark. Experientia 38: 31–35. G. Yoshida. Brune. Journal of Phycology 23: 289–296. and T. Hubbard. 2005. Wang M. Liquid fuel production using microalgae. Cultivation of microalgae in the solution from the desalting process of soy sauce waste treatment and utilization of algal biomass for ethanol fermentation. University of California. USA. 1987.E.G. Sawayama. and D. carbon utilization. Teramoto. S. Journal of Applied Phycology 11: 137–142.A. 2007. Anaerobic co-digestion of algal sludge and waste paper to produce methane. Sheng. Kunii. Colorado. Sata. Goebel. 1982..184 Diesel from Biomass Shirai F. K. 1978. Applied Microbiology and Biotechnology 54: 112–117. Ph. The ocean as a supplier of food and energy.C. K. From harmful alga to clean energy. Wilcox H. K. Wu Q. 2000. SERI/STR-2312840. Report to the Solar Energy Research Institute. and R. R. Photobioreactor design: Mixing. 1987. Golden. Dai.. Total lipid production of the green alga Nannochloropsis sp.. World Journal of Microbiology and Biotechnology 14: 839–843. Tsukahara K. California. San Diego. California. Shiraiwa. Goebel. Q11 under different nitrogen regimes. Takagi M. Berkeley. Journal of the Japan Petroleum Institute 48(5): 251–259. Bioresource Technology 98: 130–134..P. 1999. and T. Y. Murao.. Y. Nakayama. Uziel M.. G. Limited feeding of potassium nitrate for intracellular lipid and triglyceride accumulation of Nannochloris sp. J. and S. Design and analysis of pond systems for the purpose of producing fuels. Weissman J. 2007. Fu. E.S.P. J. 1998. and J. Benemann. and oxygen accumulation. Biotechnology and Bioengineering 31: 336–344. Holzer.R.C. . UTEX LB1999. C. Tornabene. Updated energy and greenhouse gas emission results of fuel ethanol. 1988. Proceedings of the 15th International Symposium on Alcohol Biofuels 26–28 September 2005.D Dissertation. and S. Focus Denmark 3: 18–19. Yen H.

Part III Ethanol and Butanol .


Improvements in Corn to Ethanol Production Technology Using Saccharomyces cerevisiae
Vijay Singh, David B. Johnston, Kent D. Rausch and M.E. Tumbleson



The corn to ethanol production industry has grown 350 % from 2000 to 2008 (RFA, 2008) due to the combination of three main factors: (i) a strong and sustained demand for ethanol as a fuel oxygenate; (ii) initially low corn prices; and (iii) initially high ethanol prices. Concomitantly, improvements in fermentation technology and process design for ethanol production have been reported. However, in 2007, corn prices were at record highs and ethanol prices were low, thus dramatically reducing ethanol plant profit margins. In turn, these margin constraints provide important corporate incentives to attain further improvements in fermentation technology or in biocatalysis and process design as a means to increase ethanol productivity, and thus restore plant profitability. In this chapter, some technologies that can potentially increase fermentation efficiency and increase final ethanol concentration are highlighted.


Current Industrial Ethanol Production Technology

Wet-milling and dry-grinding are the two major processes used to convert corn grain into ethanol. In the conventional wet-milling process, corn is fractionated into its individual

Biomass to Biofuels: Strategies for Global Industries Edited by Alain Verts, Nasib Qureshi, Hans Blaschek e and Hideaki Yukawa The contribution of Johnston has been written in the course of his official duties as US government employee and is classified as a US Government Work, which is in the public domain in the United States of America.


Ethanol and Butanol

components of starch, protein, fiber, germ and solubles (Johnston and May, 2003). Starch is further processed to produce ethanol. However, in a conventional dry-grind process there is no fractionation; the whole corn kernel is ground, mixed with water, and processed for ethanol production (Maisch, 2003). Unlike the wet-milling process, which is designed to make optimal use of the corn grain, the dry-grind process is designed to optimize process economics. As a result, during the past 15 years in the US, only dry-grind ethanol production capacity has increased such that, in 2007, more than 80 % of the US ethanol was produced using the dry-grind corn process (RFA, 2008). In the dry-grind process, the pH of the ground corn slurry (27–37 % solids content) is first adjusted with anhydrous ammonia to stabilize within the range 5.5 to 6.5. The slurry is then heated to 85  C (185 F) for 30–45 min, and subsequently cooked with highpressure steam in a jet cooker at 104  C (220  F). After cooling to 85  C (185  F) and a holding period of 30–45 min, the slurry is reheated and cooked to gelatinize the starch and break down its crystalline structure. The resulting mixture of amorphous starch is called the ‘mash.’ The next step of the process is a liquefaction, which involves an enzymatic digestion of the starch molecules in the mash into individual sugar units and oligosaccharides. This is achieved using commercial preparations of alpha-amylase. Alpha-amylase is an endoenzyme that randomly hydrolyzes a-1,4-glucosidic bonds to reduce the viscosity of gelatinized starch, producing soluble dextrins and oligosaccharides. The total alpha-amylase dose typically varies from 0.2 to 0.4 kg enzyme per metric ton of dry solids (0.02–0.04 %, w/w). The common practice is to add one-third of the alphaamylase prior to cooking, and the remainder after the jet cooker step (Kelsall and Piggot, 2009). The typical carbohydrate profile of liquefied corn mash has the following composition: maltotetraose and other soluble dextrins 24–35 %; maltotriose 1.7–3.3 %; maltose 0.4–2.7 %; and glucose 0.1–1.3 %. Typically, the result is a starch hydrolysate that has a dextrose equivalent ranging from 10.4 to 19.1 (Johal and Deinhammer, 2006). (The dextrose equivalent is a measure of the percentages of glucosidic bonds that are hydrolyzed during liquefaction.) After liquefaction, the corn mash is cooled to 32  C. It is at this stage that a second enzyme, glucoamylase, is added (0.05–0.08 %, w/w). Glucoamylase is an exoenzyme that catalyzes the release of successive glucose units from the nonreducing ends of soluble dextrins. It acts by hydrolyzing both linear a-1,4-glucosidic and branched a-1,6-glucosidic linkages (Kelsall and Piggot, 2009). It is the glucose that is generated by glucoamylase during this saccharification step which is fermented into ethanol by yeasts (typically Saccharomyces cerevisiae). Yeast cells require free amino nitrogen (FAN) for their growth and maintenance. As corn mash is deficient in FAN and urea, both are added (FAN 70 mg lÀ1; urea 480–960 mg lÀ1) to enable optimal yeast growth (Ingledew, 2005). It is worth noting that glucose production (saccharification) by glucoamylase and ethanol fermentation by yeast can be conducted simultaneously. This processing mode is nowadays common industry practice, and is referred to as simultaneous saccharification and fermentation (SSF). Prior to the 1990s, saccharification was conducted more often than not separately from fermentation. However, the practitioners in the field typically observed that separate saccharification results in high glucose concentrations that cause the mash to have a high osmotic pressure, which in turn negatively affects yeast performance. Notably, osmotic stress can result in the production during the fermentation process of higher concentrations of glycerol as glycerol aids yeast with osmoadaptation; this is of course

Improvements in Corn to Ethanol Production Technology


detrimental to the overall economics of the process. As a result, and to reduce osmotic stress on yeast cells, SSF is used predominantly in all dry-grind corn facilities. The glucoamylase dose and yeast conditioning (yeast should be in their logarithmic growth phase prior to fermentor inoculation) must be coordinated so as to optimally conduct glucose production and utilization during SSF. Despite these improvements, high glucose concentrations are commonly observed in dry-grind ethanol fermentation plants during the first 20–40 h of the fermentation phase, even when SSF is employed. As expected, and as manufacturing learning curves occur, some plants are able to control the sugar concentration of the fermentation mixture better than others (Figure 9.1: plant 1 versus plant 2, respectively). The time necessary to complete a fermentation varies among plants, but a typical range is 48–72 h. After fermentation, the spent mash is referred to as ‘beer.’ Depending on the corn slurry solids contents, the final ethanol concentrations in beer after a 60 h period of
18 Sugars %w/v, Ethanol % v/v and Glycerol %w/v Concentrations 16 14 12 10 8 6 4 2 0 0 10 20 30 40 50 60 Fermentation Time (hr) Plant 1 Mash Solids 32.0% DP4+ (%w/v) DP3 (%w/v) DP2 (%w/v) Glucose (%w/v) Ethanol (%v/v) Glycerol (%w/v)

20 Sugars % w/v, Ethanol %v/v and Glycerol %w/v Concentrations 18 16 14 12 10 8 6 4 2 0 0 10 20 30 40 50 60 Fermentation Time (hr) DP4+ (%w/v) DP3 (%w/v) DP2 (%w/v) Glucose (%w/v) Ethanol (%v/v) Glycerol (%w/v) Plant 2 Mash Solids 34.0%

Figure 9.1 Mean sugar, ethanol and glycerol concentrations at two 45 million gallon per year dry-grind plants during simultaneous saccharification and fermentation (SSF). DP2: maltose; DP3: maltotriose; DP4 þ : all saccharides with four or more glucose units


Ethanol and Butanol

fermentation ranges from 14 % to 20 % (v/v) (Kelsall and Piggot, 2009). High final ethanol concentrations (i.e., > 14 %) have been routinely achieved during the past 10 years by the dry-grind ethanol industry, primarily as a result of high-gravity fermentation research conducted by Ingledew and coworkers (Thomas and Ingledew, 1990; Thomas and Ingledew, 1992; Thomas et al., 1993; Jones and Ingledew, 1994a; Thomas et al., 1996). Prior to the 1990s, typical corn slurry solids were less than 20 % and the final ethanol concentrations were 12–14 % (v/v). Low solids content (i.e., < 20 %) were used because high ethanol concentrations ( > 10 % v/v) were inhibitory to the industrial yeast strains used and would reduce the yeast growth and cell density in the mash (van Uden, 1984). These findings have since been challenged, however, and ethanol concentrations up to 23.8 % have been produced in controlled (temperature, pH and FAN level) laboratory conditions (Thomas et al., 1996) using conventional yeast. A high final ethanol concentration improves the plant profitability by increasing the plant capacity and decreasing the downstream processing costs, thereby improving plant efficiency (Lewis, 1996; Thomas et al., 1996). Higher final ethanol concentrations can be achieved simply by increasing the dry solid contents of corn slurries (or high-gravity mash). Nonetheless, at a high initial dry solids content ($33 %), it is particularly important to ensure that the nutritional needs of the yeast are appropriately met as a preventive measure to avoid stuck or sluggish fermentations. Temperature staging (33  C during early stage and 28  C or lower during later stages) is also important to complete a high-gravity mash (Jones and Ingledew, 1994c). The addition of proteases (Thomas and Ingledew, 1990; Jones and Ingledew, 1994a), yeast extracts (Jones and Ingledew, 1994b) and amino acids (Pierce, 1987; Ingledew and Patterson, 1999) have all been reported to provide useful nutrients to yeasts, thereby increasing fermentation rates and final ethanol concentrations in high-gravity mashes. Assuming a 70 % starch content in corn, a mash containing 33 % solids would have after complete hydrolysis a theoretical maximum sugar concentration of 25.6 % (w/v). The yeast S. cerevisiae is known to tolerate sugar concentrations up to 38 % (w/v) (Ingledew, 1999). The generation of a mash with a sugar concentration of 38 % (w/v) would require to use as initial corn slurry a slurry containing 48 % solids. To date, the use of such a high solids content has not been attempted in industrial ethanol plants, primarily for two reasons: (i) the slurry viscosity increases as the sugar concentration increases, therefore creating pumping problems; and (ii) the enzymatic hydrolysis becomes inefficient as the sugar concentration goes beyond the enzyme optimum. Advances in biocatalysis (e.g., by the engineering or isolation of enzymes that both reduce the viscosity and improve the efficiency of hydrolysis) on the one hand, and deepening the fundamental knowledge of yeast nutritional needs on the other hand, have allowed dry-grind plants to use as high as 37 % dry solids in corn slurries (Johal and Deinhammer, 2006). Today, technologies are being developed that will allow further increase in dry solids content, to improve ethanol productivity, and to increase dry-grind profitability. Some of these technologies include: (i) granular starch-hydrolyzing enzymes (Robertson et al., 2006; Wang et al., 2007); (ii) corn fractionation (Singh and Eckhoff, 1996; Singh et al., 1999; Wahjudi et al., 2000; Singh et al., 2005; Wang et al., 2005); (iii) vacuum fermentation (Ramalingham and Finn, 1977; Cysewski and Wilke, 1977; Shihadeh et al., 2007); and (iv) dynamic control of the SSF process (Murthy et al., 2006a; Murthy, 2007).

Improvements in Corn to Ethanol Production Technology



Granular Starch Hydrolysis

Granular starch-hydrolyzing (GSH) enzymes (e.g., StargenÔ, manufactured by Genencor International, Palo Alto, CA; or BPX, Novozymes NA, Richmond, NC) have recently been developed. These enzymes have high GSH activities and can convert starch into dextrins at temperatures lower than 48  C. Moreover, they hydrolyze dextrins into fermentable sugars during SSF (Wang et al., 2007). Notably, the use of GSH enzymes in the dry-grind process does not require heating of the corn slurry to high temperatures for cooking or liquefaction; therefore, GSH enzymes reduce the overall utility requirements of the drygrind process. As a result, when such enzymes are used the liquefaction, saccharification and fermentation steps can all be combined into one single step – that is, simultaneous liquefaction, saccharification and fermentation (SLSF). In this particular process, ground corn, water, GSH enzyme and yeast are mixed together and fermentation is carried out. The increase in viscosity of the corn slurry that occurs during gelatinization and cooking (as in the conventional process) does not happen in the GSH process. Indeed, sugar concentrations throughout the SSF remain low or negligible when GSH enzymes are used (Figure 9.2). A primary benefit is that yeast cells are thus subjected to a low osmotic stress, thus improving the overall productivity, for example by lowering the production of glycerol (as mentioned above). During SLSF, the glucose concentrations with GSH enzymes are typically lower as compared to conventional enzyme treatments, but the final ethanol concentrations and ethanol yields remain similar (Wang et al., 2007). On the other hand, glycerol concentrations are lower for GSH treatment compared to conventional enzyme treatments. Perhaps one of the main advantages of GSH enzymes is that they do not exhibit any practical maximum viscosity threshold, since heating of the slurry is not required; consequently, higher concentrations of solids can be used in corn slurries, which allows the fermentation to reach increased final ethanol concentrations. The rate of glucose production by GSH enzymes parallels the rate of glucose fermentation by yeast, thus
20 Sugars %w/v, Ethanol % v/v and Glycerol %w/v Concentrations 18 16 14 12 10 8 6 4 2 0 0 10 20 30 40 50 60 Fermentation Time (hr) DP4+ (%w/v) DP3 (%w/v) DP2 (%w/v) Glucose (%w/v) Ethanol (%v/v) Glycerol (%w/v)

Figure 9.2 Mean sugar, ethanol and glycerol concentrations with GSH enzymes during SSF. DP2: maltose; DP3: maltotriose; DP4 þ : all saccharides with four or more glucose units


Ethanol and Butanol

enabling the practitioner to maintain a low glucose concentration during SLSF. However, at present the cost of GSH enzymes is approximately double that of conventional enzymes.


Corn Fractionation

A modified dry-grind process (E-Mill) has been developed in which the germ, pericarp fiber and endosperm fiber can be recovered as coproducts in addition to ethanol and DDGS (Singh et al., 2005; Wang et al., 2005). The E-Mill process involves soaking corn kernels in water for a short period of time (6–12 h), followed by coarse grinding and incubating with protease and amylases for 2–4 h. The germ and pericarp fiber are recovered by specific gravity difference in hydrocyclones (Singh and Eckhoff, 1996; Singh et al., 1999; Wahjudi et al., 2000). The endosperm fiber can be recovered by using a variety of screens (200 mesh or 0.074 mm openings) either prior to (Singh et al., 2005) or after (Wang et al., 2005) fermentation. The recovery of endosperm fiber after fermentation is an important step as it reduces the loss of starch in the endosperm fiber fraction. The use of GSH enzymes during E-milling results in a synergy. On the one hand, germ and pericarp fiber removal is conducted by increasing the specific gravity of the slurry, but on the other hand the GSH enzymes increase the specific gravity of the slurry and facilitate separation of the germ and pericarp fiber. The observed increase in slurry specific gravity is due to an increase in soluble starch concentration produced by the action of the GSH enzymes on granular starch in the slurry. Therefore, the use of GSH enzymes improves the fractionation process, as well as converting starch into dextrins and sugars for fermentation. Removal of the suspended solids improves both the fermentation rate and ethanol productivity (Thomas et al., 1996), because such solids interfere with the enzyme kinetics, heat transfer and mixing of the mash. When Singh et al. 2005 compared the fermentation profiles of the E-Mill and conventional dry-grind processes (Figure 9.3), the final ethanol
14 Ethanol Concentration %v/v 12 10 8 6 4 2 0 0 20 40 60 80 Fermentation Time (hr)
Conventional E-Mill Conventional Rate E-Mill Rate

Figure 9.3 Comparison of ethanol concentrations and fermentation rates between conventional and E-Mill dry-grind processes. Fermentation was 100% complete in the E-Mill process after 36 h, but only 64% complete in the conventional process. The final ethanol concentration in the E-Mill process was 27% higher than for the conventional process. Reproduced from V. Singh et al. 2005, with permission from AACC

Improvements in Corn to Ethanol Production Technology


concentration was increased by 27 % when nonfermentable materials (e.g., as germ, pericarp fiber and endosperm fiber) were removed from the mash and their volumes replaced with fermentable substrates (e.g., corn). The fermentation rate of the E-Mill process is twice that of a conventional dry-grind corn process (Figure 9.3), with the E-Mill fermentation typically being complete at 36 h but the conventional dry-grind corn process requiring 72 h. This completion time is a critical parameter, as a faster turn-around of batches will allow a dramatic increase in overall plant productivity. Corn fractionation, especially the removal of germ and pericarp fiber, can be conducted on raw corn (without any soaking step) using conventional dry-milling equipment. However, dry corn fractionation results in a loss of nutrients (water-soluble proteins) from the germ fraction. In the E-Mill process, these proteins are transferred into the water during the soaking step, and are available to yeast as nutrients during fermentation, whereas the dry separation of germ from corn endosperm with dry-milling equipment results in a loss of starch from the germ fraction. Murthy et al. 2006a compared dry and wet fractionation prior to ethanol production and reported lower fermentation rates and final ethanol concentrations of dry-fractionated corn.


Simultaneous SSF and Distillation

Ramalingham and Finn (1977) and Cysewski and Wilke (1977) evaluated the effect of applying a vacuum during the fermentation of glucose solutions for achieving the in situ removal of ethanol. Interestingly, these authors reported a 12-fold higher ethanol productivity as compared to control treatments processed in the absence of a vacuum. Later, Arsenyev et al. (2002) attempted to combine vacuum distillation with a typical SSF process for wheat mashes. In this latter process the fermentation was conducted under vacuum, allowing the ethanol to boil off (distill) at 32  C as it was produced by the yeast. By using such continuous distillation, the ethanol concentration was maintained as negligible during the entire fermentation process. This was a clear advantage as the fermenting microorganisms were not then subjected to alcohol-mediated toxicity. Combining GSH enzymes and SSF under a vacuum allows the integration of all four unit operations (liquefaction, saccharification, fermentation and distillation) into one single step, so that the process allows for simultaneous liquefaction, saccharification, fermentation and distillation (SLSFD). Remarkably, this unique combination of unit operations eliminates both substrate (glucose) and product (ethanol) inhibition of the yeast. With the SLSFD process, higher slurry solids (40 %) and low concentrations of sugar and ethanol are possible during SSF. Due to the higher slurry solids and removal of water during vacuum distillation, the percentage of stillage solids at the end of the fermentation/SLSFD is higher, and these can be sold as wet grains without any need for centrifugation to remove water. This process also can eliminate the need to perform thin stillage evaporation. For example, Shihadeh et al. (2007) evaluated the SLSFD process for dry-grind production with 40 % slurry solids and compared it with a conventional process (Figure 9.4). Under an SLSFD process, the slurry was fermented leaving only a negligible residual glucose content. By comparison, the conventional process residual sugar in beer typically increased at 20 h, such that a final residual sugar concentration of 5 % (w/v) was observed. Overall, the ethanol productivity achieved using an SLSFD process is 20–40 % higher than that of the conventional process.


Ethanol and Butanol
20 18 16 14 12 10 8 6 4 2 0 0 20 40 60 80 Fermentation Time (hr) Ethanol % v/v and Glucose % w/v Concentrations

Mash Solids 40.0%

Glucose Vac Ethanol Vac Glucose No Vac Ethanol No Vac

Figure 9.4 Ethanol and glucose concentrations with and without vacuum fermentation. For no vacuum treatment, the ethanol concentration reaches 18% but does not increase further due to yeast inhibition. The sugar concentration starts to increase after 10 h for no vacuum treatment. For vacuum treatment, as the ethanol concentration exceeds 14%, the vacuum is applied and ethanol is stripped. There is no build-up of sugars during fermentation, and the yeast is able to ferment all sugars into ethanol. Courtesy of Jameel Shihadeh


Dynamic Control of SSF Processes

In dry-grind production, only three in-process parameters – the pH, temperature and glucoamylase dose – are monitored or controlled during SSF. In most plants, only the temperature is controlled, whereas the pH is adjusted prior to SSF and not controlled during the fermentation. Similarly, the glucoamylase dose is fixed, with the glucoamylase solution being pumped at a fixed rate during the first 15–20 h of SSF. The setting for these process parameters is based on a compromise between the optimum conditions for enzymatic hydrolysis and yeast metabolism. SSF is a microbial and biologically dynamic process, since the fermentation medium chemistry changes as the fermentation proceeds, causing changes to the physiological responses of the fermenting microorganisms. In many manufacturing plants, process controllers are used to maintain the temperature during SSF and adjust the pH and enzyme pump rate prior to or at the very start of SSF via ‘fixed’ control strategies. Whilst such strategies have the advantage of being simple, they do not respond optimally to the changing conditions of a fermentation tank. A continuous optimization of the fermentation conditions can cause dramatic improvements in fermentor productivity by providing an organism with the most appropriate environmental conditions for ethanol production. However, the design of optimal control strategies requires the use of finely tuned microbial growth models that incorporate microbial responses to changing environmental conditions. In this respect, the modeling of SSF based on yeast metabolism alone is not adequate, because enzymatic hydrolysis (dextrins to glucose, by glucoamylase) occurs simultaneously. In order to resolve this problem and to develop an optimal method for controlling SSF, Murthy 2007 simulated starch hydrolysis using a Monte Carlo simulation technique on a starch structure, and combined it with a flux balance analysis (FBA) and cybernetic model for yeast metabolism. The resultant FBA model that was established for baker’s yeast

Improvements in Corn to Ethanol Production Technology
Ethanol Concentration (% v/v)


No Dyn Control Dyn Control



8-Apr Time



Figure 9.5 Testing of dynamic controller in a commercial dry-grind plant during SSF. Open squares ¼ ethanol concentrations using a dynamic controller; solid triangles ¼ ethanol concentrations without using a dynamic controller

provided steady-state flux rates calculations for various metabolic network reactions which were, in turn, used to estimate cybernetic model parameters. The results of such yeast metabolism simulations, when conducted over a range of temperatures, pHs, organic acid concentration, initial yeast inoculum levels (Murthy et al., 2006b) and initial glucose concentrations, were found to be in good agreement with previously reported data. The optimal control of the dry-grind SSF process results in reduced fermentor glucose concentrations, typically less than 2.0 % (w/v). Compared to the standard SSF process, the use of an optimal controller results in 50 % reduction in enzyme (glucoamylase) use under varying operating conditions (Murthy, 2007). When the as-yet developed optimal controller was tested in a commercial dry-grind ethanol plant, although a 35 % reduction in glucoamylase dose was observed compared to control treatment, the final ethanol concentrations were similar for the two systems (Figure 9.5) (Murthy, 2007).


Cost of Ethanol

Kwiatkowski et al. 2006 developed a detailed process and cost model of a conventional dry-grind ethanol plant, to estimate the cost of ethanol production. The model incorporated the composition of raw materials and products, major unit operations, utilities, capital and operating cost estimation, product and coproduct revenues. The unit cost of ethanol production (minus any coproduct credit) was estimated as US$ 1.04 per gallon (US$ 0.27 lÀ1). In this case, over 75 % of the unit ethanol production cost was due to the corn, while over 25 % was due to utilities costs. [In this model, a corn price of US$ 2.20 per bushel (US$ 0.086 kgÀ1) was used.] The unit ethanol production cost was found to be sensitive to the price of corn. Indeed, a sensitivity analysis conducted on corn price (from US$ 2 to US$ 5 per bushel) resulted in a more than 52 % increase in unit ethanol production cost. A total capital cost of US$ 47.6 million is required for a 40 million gallons per year dry-grind ethanol plant (Kwiatkowski et al., 2006). Several studies/surveys have been


Ethanol and Butanol

conducted to determine the net operating costs of corn to ethanol production in the US, with values ranging from US$ 0.92 to US$ 2.16 per gallon (Perrin et al., 2009). The evolution of ethanol cost has been surveyed particularly in Brazil (where ethanol is produced from sugarcane) and the US (ethanol from corn) (Goldemberg et al., 2004; Shapouri and Gallagher, 2005). Over the years, a significant fall in ethanol production costs has been observed for both corn and sugarcane as these technologies have benefited from the experience gained. Nonetheless, with advancing technologies in feedstock, process engineering and coproduct development, further reductions in the unit cost of ethanol production are expected.



While corn fermentation using Saccharomyces cerevisiae to produce fuel ethanol has been used commercially for more than a decade, present-day technologies will undoubtedly contribute to improving the long-term sustainability and profitability of the corn-to-ethanol industry. Incremental innovation to improve process controls is just one example of the methods used to drive the overall process economics. The general importance of economies of learning and/or manufacturing learning curve effects is best exemplified by the achievements of the ethanol industry in Brazil, where an over 70 % decrease in the cost of ethanol production was observed between 1980 and 2002 (Goldemberg et al., 2004). In the US, Shapouri and Gallagher (2005) reported that the cost of building a new ethanol plant had decreased between 1998 and 2002, due to improved plant designs and economies of scale. Likewise, an increase in fundamental knowledge of the physiology of fermenting microorganisms, and of complementary enzymatic reactions, is critical not only to improve ethanol plant productivity but also to reduce energy input during ethanol production. Looking into the future, when cellulosic ethanol becomes industrially viable, existing corn-to-ethanol plants could be retrofitted to the new process. Indeed, some technology providers in the US have already considered integrating cellulosic ethanol production with grain ethanol production, so as to exploit synergies between the two technologies. It is also likely that some ethanol plants will be retrofitted to produce other fermentation biochemicals or fuels.

D.V. Arsenyev, A.V. Kuzmichev, A.V. Ezhokov A.A. Ezhkov and Y.Y. Pekarev. Direct distillation technology processing grain into ethanol and animal feeds. Abstract, p. 70. Proceedings of 2nd International Conference on Biotechnology and Business, Moscow, Russia, 2002. G.R. Cysewski and C.R. Wilke. Process design and economic studies of alternative fermentation methods for the production of ethanol. Biotechnology and Bioengineering 20, 1421–1444 (1978). J. Goldemberg, S.T. Coelhi, P.M. Nastari and O. Lucon. Ethanol learning curve – the Brazilian experience. Biomass and Bioenergy 26, 301–304 (2004). W.M. Ingledew. Alcohol production by Saccharomyces cerevisiae: a yeast primer, in Alcohol Textbook, 3rd ed. Nottingham University Press, Nottingham, UK, 1999, pp. 49–87. W.M. Ingledew. Improvements in alcohol technology through advancement in fermentation technology. Getreidetechnologie 59, 308–311 (2005).

Improvements in Corn to Ethanol Production Technology


W.M. Ingledew and C.A. Patterson. Effect of nitrogen source and concentration on the uptake of peptides by a lager yeast in continuous culture. Journal of American Society of Brewing Chemists 57, 9–17 (1999). M. Johal and R.S. Deinhammer. Characterization of industrial corn mashes and relationship to glucoamylase performance. Proceedings of Corn Utilization Technology Conference, NCGA, Dallas, TX, 2006, pp. 57–61 A.M. Jones and W.M. Ingledew. Fuel alcohol production: assessment of selected commercial proteases for very high gravity wheat mash fermentation. Enzymes and Microbial Technology 16, 683–687 (1994a). A.M. Jones and W.M. Ingledew. Fermentation of very high gravity wheat mash prepared using fresh yeast autolysate. Bioresource Technology 50, 97–101 (1994b). A.M. Jones and W.M. Ingledew. Fuel alcohol production: optimization of temperature for efficient very-high-gravity fermentation. Applied and Environmental Microbiology 60, 1048–1051 (1994c). J.R. Kwiatkowski, A.J. McAloon, F. Taylor, and D.B. Johnston. Modeling the process and costs of fuel ethanol production by the corn dry-grind process. Industrial Crops and Products 23, 288–296 (2006). D.R. Kelsall and R. Piggot. Grain milling and cooking for alcohol production, in Alcohol Textbook, 5th ed. Nottingham University Press, Nottingham, UK, 2009, pp. 161–175. S.M. Lewis. Fermentation alcohol, in: Industrial Enzymology, 2nd ed. T. Godfrey and S. West (eds), Macmillan Press Ltd, London, UK, 2006. W.F. Maisch. Fermentation processes and products in Corn: Chemistry and Technology, 2nd ed. P.J. White and L.A. Johnson (eds), American Association of Cereal Chemists, St. Paul, MN, 2003, pp. 695–721. G.S. Murthy. Development of a controller for fermentation in the dry-grind corn process. PhD. Thesis, University of Illinois at Urbana-Champaign, Urbana, IL, 2007. G.S. Murthy, V. Singh, D.B. Johnston, K.D. Rausch, and M.E. Tumbleson. Evaluation and strategies to improve fermentation characteristics of modified dry grind corn processes. Cereal Chemistry 83, 455–459 (2006a). G.S. Murthy, V. Singh, J.V. Medanic, K.D. Rausch, D.B. Johnston, and M.E. Tumbleson. Dynamic control of the fermentation process in dry grind corn processing. Abstract. Proceedings of Corn Utilization Technology Conference, p. 155. NCGA, Dallas, TX, 2006b. G.S. Murthy, V. Singh, J.V. Medanic, K.D. Rausch, D.B. Johnston, and M.E. Tumbleson. Dynamic control of the fermentation process in dry grind corn processing. Abstract. Proceedings of International Starch Technology Conference, University of Illinois, Urbana, IL, 2006c. P.K. Perrin, N.F. Fretes, and J.P. Sesmero. Efficiency in Midwest US corn ethanol plants: a plant survey. Energy Policy 37, 1309–1316 (2009). J.S. Pierce. The role of nitrogen in brewing. Journal of the Institute of Brewing 93, 378–381 (1987). A. Ramalingham and R.K. Finn. The Vacuferm process: a new approach to fermentation alcohol. Biotechnology and Bioengineering 19, 583–589 (1977). RFA. Renewable fuels outlook 2008. Available online at: outlook/RFA_Outlook_2008.pdf Renewable Fuels Association, Washington, DC, 2008. G.H. Robertson, D.W.S. Wong, C.C. Lee, K. Wagschal, M.R. Smith, and W.J. Orts. Native or raw starch digestion: a key step in energy efficient biorefining of grain. Journal of Agricultural and Food Chemistry 54, 353–365 (2006). H. Shapouri and P. Gallagher. USDA’s 2002 ethanol cost-of-production survey. Agriculture Economic Report No. 841, US Department of Agriculture, 2005. J.K. Shihadeh, K.D. Rausch, M.E. Tumbleson, and V. Singh. Vacuum fermentation for in situ removal of ethanol during simultaneous liquefaction, saccharification, and fermentation. AACC International annual meeting. San Antonio, TX, 2007. V. Singh and S.R. Eckhoff. Effect of soak time, soak temperature and lactic acid on germ recovery parameters. Cereal Chemistry 73, 716–720 (1996). V. Singh, D.B. Johnston, K. Naidu, K.D. Rausch, R.L. Belyea, and M.E. Tumbleson. Comparison of modified dry grind corn processes for fermentation characteristics and DDGS composition. Cereal Chemistry 82, 187–190 (2005).

A.D. Thomas.D.R.C. L. 734–738 (2005). McAloon. Process Biochemistry 31. Thomas and W. Xue. Applied Biochemistry and Biotechnology 43. Ingledew. Tumbleson. 2046–2050 (1990).M. Rausch. L. and W. P. 263–272 (1984). and K. Eckhoff. Doner. Johnston. Hynes.D. Cereal Chemistry 82. Singh.M. Moreau. Buriak. Hicks. Cereal Chemistry 76. L. P. D. and M. Wahjudi. Jones.W. H. Rausch. . Cereal Chemistry 84. Journal of Industrial Microbiology 10. and W. dry solids and residual germ on fiber yield and purity.B. Tumbleson. Singh. K. S.B. Thomas and W. K. Eckhoff. S. Effect of ethanol on the temperature relations of viability and growth in yeast. Tumbleson. 211–226 (1993).A. and M. Fuel alcohol production: effect of free amino nitrogen on fermentation of very-high-gravity wheat mashes.C.E.B. van Uden. Wang. K.C.M. Xu. Hynes. Rausch. 10–14 (2007). Quick fiber process: effect of mash temperature. Xu. V. Wang. Applied Environmental Microbiology 56. Cereal Chemistry 77. Comparison of raw starch hydrolyzing enzyme with conventional liquefaction and saccharification enzymes in dry grind corn processing. Production of fuel alcohol from wheat by VHG technology: effect of sugar concentration and temperature. J.E. Ingledew. Production of 21 % (v/v) ethanol by fermentation of very high gravity (VHG) wheat mashes. Ingledew.C. 321–331 (1996). 61–68 (1992).R. K. Ingledew.M. V.H.E. 640–644 (2000). K. D. V.H. Wang.198 Ethanol and Butanol V. A. Recovery of fiber in the corn drygrind ethanol process: a feedstock for valuable co-products. 868–872 (1999). Singh. R.M. M. Thomas. Singh. Practical and theoretical considerations in the production of high concentration of alcohol by fermentation.J. Johnston. K. P. K. and S. Comparison of enzymatic (E-mill) and conventional dry grind corn processes using a granular starch hydrolyzing enzyme. Critical Reviews in Biotechnology 1. P. S. N.

10 Advanced Technologies for Biomass Hydrolysis and Saccharification Using Novel Enzymes Margret E. Harry J. Bayer. One of the major limitations to lignocellulosic biomass conversion is the enzymatic degradation of the complex matrix of lignocellulosic polymers that form plant cell walls. Flint and Bryan A. Although hexoses (glucose) and pentoses (primarily xylose) are the most desirable soluble sugars for fermentation. White 10. by hydrolysis into soluble sugars. Brulc. Edward A. are assembled into a complex molecular machine referred to Biomass to Biofuels: Strategies for Global Industries and Hideaki Yukawa Ó 2010 John Wiley & Sons. Moreover. Nasib Qureshi. Raphael Lamed. In this regard. Hans Blaschek ` . which will release hexoses and pentoses for secondary fermentation. Plant cell wall degradation by bacteria and fungi is coordinated by a multitude of enzymes which. in many cases.1 Introduction Lignocellulosic biomass is high in cellulose and noncellulosic structural polysaccharides (xylan) which. given the expected diversity in biomass feedstock sources. Ltd Edited by Alain Vertes. Jennifer M. the most successful bioconversion processes for the production of biofuels from lignocellulose might well come from the concerted action of cellulolytic microbes that have an extensive set of glycoside hydrolases for deconstruction of the plant cell wall. can then be fermented for conversion to liquid biofuels. Berg Miller. a range of lignocellulosic substrates must be efficiently degraded by processes optimized for economically viable biofuels production. they are incorporated within the structural portion of the plant cell wall. as these are refractory to hydrolysis and degradation.

. Many side-chain constituents. Bayer et al.. ferulolyl. Thus. Details of glycoside hydrolases and cellulosomes. Thus. 1994. we will discuss the current status of cellulases and cellulosomes research.1). 2000). acetyl. and how novel enzymes might be discovered for biomass conversion to biofuels. cellulose is found in two forms. 1998). presumably due to the coevolution of microbes and plant cell wall types. which include both cellulases and noncellulosic structural polysaccharidases. and their various families.. Within these microfibrils. Other noncellulosic structural polysaccharides. including arabinofuranosyl... which is made up of b-1. are modular enzymes. or galactoglucomannan. Bayer et al. Noncellulosic structural polysaccharides are branched heteropolysaccharides that are associated with cellulose and lignin in the plant cell wall.4-linked-D-glucopyranose and b-Dmannopyranose units with a-1. 2004). modular and subunit architectures. branch off the main backbone. and methylglucuronyl groups.. although the chemical complexity of noncellulosic compounds is much greater than that of cellulose (see Figure 10. there are many possible routes to optimizing the enzymatic breakdown of lignocellulosic materials. 2000. namely amorphous and crystalline. Bayer et al. 1998.4-linked xylopyranose units. are also commonly found in the plant cell wall. Bayer et al. wherein each glucose unit is rotated 180 relative to its neighbor (Beguin and Aubert. 1994.200 Ethanol and Butanol as the cellulosome (Bayer et al. this allows the cellulose microfibrils to be embedded within a matrix of noncellulosic structural polysaccharides (Bayer et al.. 10. the repeating subunit in cellulose is actually cellobiose.2 The Substrate Cellulose is a linear polymer made up of repeating units of glucose linked by b-1. the rumen provides a unique genetic resource for the discovery of plant cell wall-degrading microbial enzymes for use in biofuel production.. Therefore. The search for novel enzymes and future application to biomass conversion are discussed. 2000). 10. 2000). 1998. and novel molecular approaches discussed that might lead to an efficient process for the conversion of lignocellulosic biomass to soluble sugar components for biofuel production. One approach is based on the observation that the rumen habitat contains a very efficient consortium of microbes that harbor a complex lignocellulosic degradation system for the microbial attachment and digestion of plant biomass.1). which can then be assembled into plant cell walls (Beguin and Aubert. The individual cellulose chains are then tightly packed and organized in parallel into crystalline microfibrils. the structural rigidity is much less. Bayer et al.. are presented.4 glucosidic bonds (Figure 10.3 Glycosyl Hydrolases Glycosyl hydrolases (GHs). such as arabinogalactan or lichenins.6 galactose residues (Bayer et al. These modules consist of different combinations . In this chapter. They are typically composed of main-chain backbones of xylan. However. which consists of b-1. 2000). The crystalline form of cellulose is particularly difficult to degrade and typically makes up the core of a cellulose microfibril (Bayer et al. based on its structural characteristics.

Advanced Technologies for Biomass Hydrolysis HO O O OH HO O OH O O O CH 3 O OH OH 201 Cellulose OH O HO OH O HO O OH HO O OH O OH O O HO O HO OH O O OH O O OH O O O OH O Acetyl Xylan O OH O OH OH O O HO OH O H 3C H3C HO HO OH O O HO OH O O OH O HO HO OH Cellobiose Diferulic Acid Bridge Xylobiose OH HO OH O OH O OH O HO O OH O O O Arabinose O O O O OH Xylan O O HO O HO O O OH O O OH O OH Ferulic Acid O H3C O ρ-Coumaric Acid O O CORE LIGNIN Figure 10.1 Chemical structures of cellulose and noncellulosic structural polysaccharides found in the plant cell wall .

The two main mechanisms involve either the retention or inversion of the anomeric carbon configuration. 48. A water molecule then hydrolyzes the bond between the substrate and nucleophile to form a product that has the same stereochemistry as the original substrate. 45. while arabinofuranosidases. while others need the cellulose to be partially degraded before they can utilize it. There are enzymes that are specific for either the reducing or nonreducing end of the cellulose chain. such as that found in a plant cell wall. is that many GHs act on several different substrates. which fall into 12 clans designated A–N. Both mechanisms require a pair of carboxylic acid residues. The retaining mechanism is a double-displacement mechanism that involves the formation of a glycosyl–enzyme intermediate. For cellulolytic organisms. which can then be confirmed with a three-dimensional (3-D) crystal structure (Henrissat and Davies. Still other enzymes need the cellulose to be at the end stages of degradation when it is no longer in its crystalline state (Bayer et al. glucuronidases. 2000). Noncellulosic structural polysaccharidases are a diverse group of enzymes. In addition. they have perhaps evolved due to the complexity of the substrate. Moreover.. In this mechanism. within the active site (McCarter and Withers. cleave the b-1. The base activates the water molecule so that it can attack the . namely 5. Bayer et al.. 2000).org].cazy.. Accessory modules can include carbohydrate-binding modules (CBMs). 7. 1998. Bayer et al. there are also many other modules. GHs are classified into families and clans based on amino acid sequence similarity. the functions of which remain unknown (Bayer et al. 8. there are 112 families of GHs. the structural features of these enzymes are not taken into account by substrate-based methods (Henrissat and Davies. The most current sequence-based classifications are described on the Carbohydrate Active Enzymes database (CAZy) [http://www. some enzymes have specificities for cellulose at varying degrees of crystallinity. There are also enzymes that prefer cellulose in its crystalline form before degradation has really begun. found in GH families. Degradation of the main chain is usually performed by xylanases and mannanases. xylosidases and mannosidases degrade the side-chain constituents (Bayer et al. sortase motifs or cell-adherence modules. a variety of cellulases are required. and the other acting as a general base. one residue acts as an acid/base catalyst to protonate the glycosidic oxygen. resulting primarily in the production of cellobiose.4-glucosidic bonds of cellulose. 1997). The inverting mechanism proceeds by a direct displacement of the leaving group by water along with two residues: one acting as a general acid. mechanism and evolutionary relationship of a GH. these enzymes are very important for the degradation of plant cell walls because they remove the structures surrounding the cellulose. dockerins. 1994).. These include enzymes that can cleave many different types of bonds. such as Ruminococcus flavefaciens. The ‘true’ cellulases. 9. and those that degrade side-chain constituents. The reason for using a sequencebased classification. According to the most recent release of this database.. 6. Using a sequence-based classification. Glycoside hydrolases use a general acid catalysis to hydrolyze the glycosidic bond. 1997. rather than traditional methods that focus on the substrate and the bond that is being cleaved. In order to fully degrade crystalline cellulose. 2000).202 Ethanol and Butanol of catalytic domains with accessory or ‘helper’ modules. such as a glutamate or an aspartate. while the other residue acts as a nucleophile and forms the glycosyl–enzyme intermediate. and 74. These enzymes fall into two major categories: those that degrade the main-chain backbone. acetyl esterases. thereby allowing organisms to access the cellulose needed for their growth and metabolism. 2000). one can reflect the structure.

or at the ends of the polysaccharide chain. 1998. that have both endo and exo characteristics (Gal et al. 2000). Efficient cellulose degradation by anaerobic bacteria is achieved by relatively small quantities of enzymes they produce. The endo-acting enzymes. the distance between the catalytic residues of  inverting enzymes is larger ($10 A) than that of retaining enzymes ($5. 1998.5 A) (McCarter and Withers. 1993. Processivity is a common characteristic of enzymes containing a GH9 closely linked to a CBM3c. cellulolyticum has the same GH9/CBM3c modular composition. respectively. and has been reported to exhibit the same type of activity as the enzyme from T. is tunnel-shaped such that the polysaccharide chains are threaded through the active site in order to cleave the ends (Gilbert and Hazlewood. which ultimately results in a stereochemistry opposite to that of the original anomeric carbon conformation. Beguin and Aubert. but outperformed Cel5A as the crystallinity of the substrate was increased. These types of enzymes are often said to have the characteristic of ‘processivity’ (Davies and Henrissat. The structure of one such enzyme from Thermomonospora fusca revealed that the CBM is in line with the active site of the GH9 catalytic domain such that they can interact. 10. similar to an endoglucanase. however. Bayer et al.. Bayer et al.4 The Cellulosome Concept Fiber degradation is coordinated by a multitude of bacterial and fungal GHs. When Cel9G was compared to the endoglucanase. This particular CBM is also unique in that. but they also illustrate how accessory modules can contribute to the intricacy of GHs and modify their mode of action. such as exoglucanases or cellobiohydrolases. for instance.. 1997.. Often. Cel5A. which allows them to straddle the substrate anywhere along the polysaccharide chain. 1994. has two such enzymes. Davies and Henrissat. A processive enzyme cleaves bonds anywhere along the cellulose chain. The endo. have a cleft. 2000). The active site of exo-acting enzymes. Glycoside hydrolases are often categorized as being either endo-acting or exo-acting enzymes.mode of action refers to whether the enzyme can cleave bonds within the polysaccharide chain. As a result. Clostridium cellulolyticum. compared to aerobic microorganisms. Reverbel-Leroy et al. rather than binding tightly to the cellulose surface.. it most likely is involved in disrupting the cellulose structure in order to feed the cellulose substrate through the active site of the GH9 (Sakon et al.. 1997). The CBM3c of Cel9G was also found to be essential for catalytic activity because the GH9 alone could not hydrolyze any of the cellulosic substrates (Gal et al.. such as endoglucanases. the distinction between these enzymes can be unclear because there are some enzymes that have both endo and exo activity (Bayer et al. Bayer et al.’ similar to the activity seen in an exoglucanase.and exo. 1994. indicating that it was able to access more sites for hydrolysis (Gal et al. make it difficult to define the action of the enzyme. The processive endoglucanase (Cel9G) from C.. Cel9G showed the same mode of endo-action as Cel5A. there needs to be more space between the catalytic residues to accommodate both the substrate and water. and then continues along the substrate subsequently cleaving off the ends of the cellulose chain ‘processively. Bayer et al. This enigma was clarified in .. such as these.. 1997).Advanced Technologies for Biomass Hydrolysis 203 protonated glycosidic oxygen. Cel48F and Cel9G. The structure of these enzyme types also distinguishes this behavior. As the water molecule is directly involved in this reaction. 1997). 1995. 2000). fusca. 1995).. Processive enzymes. 1997).or groove-shaped active site.

. OlpB and Orf2p) encoded by the sdbA (scaffoldin dockerin binding) gene (Leibovitz and Beguin. The scaffoldin in this particular cellulosome contains a carbohydrate-binding module (CBM). OlpA. Bayer et al. E.. 1997). Since the establishment of the cellulosome concept (Lamed et al.2 Schematic of the cellulosome of Clostridium thermocellum. which includes catalytic modules. The ‘type II’ dockerin of the C. These proteins also characteristically possess a modular architecture. which contains the cohesin domains that interact with the dockerin modules of the enzymatic subunits.. in C. 1996. 2000). 1983). The various enzyme subunits act synergistically to hydrolyze cellulose and other plant polysaccharides. K. M. Gerngross . has been derived from the study of Clostridium thermocellum (see Figure 10. the molecular details of various scaffoldin proteins are only now beginning to emerge (Shoseyov et al.204 Ethanol and Butanol Figure 10. Cellulosomes primarily reside within cell-surface protuberances and the culture medium. 1998.2. which possesses a series of functional domains involved in enzyme attachment (cohesins). thermocellum scaffoldin binds to a type II cohesin which.. and a module (dockerin) supporting interaction with the cohesin-bearing scaffoldin. The former is commonly referred to as the scaffoldin or cellulosome-integrating protein (CipA). and the complex is attached to the cell surface via an anchoring scaffoldin containing an S-layer homology domain and a single cohesin that interacts with a dockerin on the main scaffoldin (Bayer et al. 1997). Bayer et al.g. The cellulosome components include the scaffoldin subunit. SdbA. Leibovitz et al. Stackebrandt.. Falkow.. Although the existence of cellulosomes in a wide variety of different anaerobic bacteria has long been established (Lamed et al. 1983).. is present in surface-anchoring proteins (e. Schleifer. a CBM and anchoring to the bacterial cell surface. The ‘type I’ dockerins of the catalytic proteins interact selectively and in a species-specific manner with the type I cohesins of the scaffoldin (Pages et al. see also Bayer et al.. Cellulosomal proteins can be subdivided into noncatalytic and catalytic components. 1992.. part by the identification of the cellulosome: a multienzyme complex specialized in cellulose degradation. 2000. S. Rosenberg. architecture. 2007. 2004 for recent reviews). E. Reproduced with kind permission of Springer Science þ Business Media: The Prokaryotes 3rd edn.. much of our understanding of its catalytic components. CBMs.-H.. thermocellum.. Dworkin... and mechanisms of attachment to the bacterial cell and to cellulose.

1999). the intractability of virtually all specialist cellulose-degrading bacteria to genetic manipulation has precluded a thorough dissection of their lignocellulose degradation systems. neither exo-acting nor processive endocellulases typical of those required for cellulose degradation in other microbes are found in its genome (Qi et al. Genome sequencing has been used to reveal the relevant molecular components for cellulose degradation in these ruminal microorganisms via sequence similarities to CAZyme components from other organisms. and they derive nutrition from plant cell walls via the enzymatic action of anaerobic microbes present in the rumen.1 Comparative Genomics There are three major specialist lignocellulose-degrading bacteria in the rumen. It is believed that evolution has favored the cellulosome as the preferred mode of enzyme organization in fibrolytic. Interestingly.. there appear to be three different paradigms for lignocellulose-degradation based on the genome projects which have been coordinated by the North American Consortium for the Genomics of Fibrolytic Ruminal Bacteria (http://www. 1994. It is anticipated that the approaches described below will identify thousands of novel cellulases and cellulosome components to use as a basis for the engineering and optimization of protein function to enhance the degradative activity on grass-derived lignocellulosic substrates. the greatest lignocellulose-degrading activity on grass-derived substrates is found in grazing ruminant cattle.. One strategy to overcome this is based on the premise that molecular engineering will be most successful when starting from the most active naturally occurring systems. The carbohydrate-active enzyme systems (CAZymes) of rumen bacteria provide a starting point for discovering. 2005. while very efficient. in terms of the numbers and evolutionary origins of the genes encoding for this function. However.. with the exception of C... 1997). succinogenes is unique in that while this bacterium harbors an abundance of cellulase genes. especially for other cellulosome-producing bacteria. funded by the USDA). 10... In nature. Pages et al. Cattle eat grass. In addition. Pohlschroder et al. Although much is known about the structure–function relationships of numerous GHs. much remains to be learned about the extent to which cellulosome composition and location are affected by factors such as polysaccharide complexity. anaerobic bacteria and fungi. F. little information is available regarding the mode of cell-surface attachment of other cellulosome-producing bacteria. is distinctly different from the other two bacteria. and Ruminococcus flavefaciens (Forsberg et Kakiuchi et al. Each of these bacteria has evolved an organism-specific mode of plant cell wall deconstruction which. Ruminococcus albus. Pohlschroder et al. 1985. . There is also a limited amount of information that suggests that the composition and disposition of the cellulosome can be affected by carbon source (Bayer et al. Fibrobacter succinogenes.tigr. 1998. characterizing.Advanced Technologies for Biomass Hydrolysis 205 et al..5. 1989. and ultimately engineering lignocellulose-degrading systems customized for the deconstruction of biomass substrates.5 New Approaches for the Identification of Novel Glycoside Hydrolases There are many possible routes to optimizing the enzymatic breakdown of lignocellulosic materials. thermocellum. Miron et al. 1993.. 1995). 10.

Most recently. 1998. J. the scaffolding protein ScaA incorporates a group of dockerin-containing enzymes into its three resident cohesin repeats (Rincon et al. ScaA. R. The R. both proteins possess a single copy of a novel ‘X’ module. Navarre and Schneewind. 2008). Rincon et al. there is no evidence in the genome sequence for a cellulosomelike structure. 2001. flavefaciens cellulosome differs from the previously defined mode of cellulosome attachment. R. 2003).. 2001). on the other hand. recently shown to be a unique type of carbohydrate-binding module (CBM37. Taken together. In addition. In R. 1997).. 2000.. The proposed sortasemediated attachment of the R. 2003). albus possesses novel features underpinning its ability to degrade plant fiber.. It thus appears that the R. Neither Cel9B nor Cel48A contains a dockerin. albus 8 and three mutant strains defective in their adhesion to. 1994. which suggests that it is positioned covalently on the cell surface via proteolytic cleavage and transfer to the cell wall in a well-documented sortase-mediated attachment mechanism common in Gram-positive bacteria (Navarre and Schneewind. albus glycoside hydrolases. Instead. a small ScaC scaffoldin serves as an adaptor protein that enhances the repertoire of cellulosomal subunits by binding both ScaA via its dockerin and to a plethora of as-yet unidentified polypeptides via its single divergent cohesin (Rincon et al. albus possesses a number of other Pil gene homologues (Pegden et al. Rincon et al. cellulose (Devillard et al. Rincon et al. Ton-That and Schneewind. mass spectrometry and genome sequencing have been used to examine proteomic differences among R. via anchoring proteins that contain an S-layer homology module (Leibovitz et al. Moreover. Currently. ScaE includes an N-terminal cohesin and a C-terminal LPXTG-like motif. . and degradation of... 2001. ScaB and ScaC. Rakotoarivonina et al. 2005).. albus draft genome (8 Â coverage. that is. Craig Venter Institute). 2001. Xu et al. 2004).... 2004. 2005). 2004).. 2004). Data acquired using two-dimensional polyacrylamide gel electrophoresis.. ScaE.. Rincon et al. In addition to the fimbrial homologue implicated in adhesion (CbpC). There is also an absence of transposable elements in this genome. Morrison. has supported several key advances in our understanding of fiber degradation by this bacterium.. together with interacting enzymes and other unidentified dockerin-bearing proteins (Aurilia et al. encoded by the sca gene cluster. The assembly of these components differs markedly from the proposed molecular architecture in the Clostridial cellulosomes.. Schneewind et al. research on Ruminococcus flavefaciens 17 has revealed a cellulosome complex comprising numerous cohesin-containing scaffoldins. 2003. and ScaB is attached to the cell surface with the latter complement of scaffoldins and enzymes (Ding et al. Rincon et al. In turn... flavefaciens cellulosome is attached to the cell surface via this novel ‘XDoc-Coh’ ScaB–ScaE interaction. an additional component. 1999. has been discovered that interacts with an X-module together with an atypical dockerin of ScaB (Rincon et al. which suggests that their retention on the bacterial cell surface and recovery by cellulose affinity procedures does not involve a Clostridial-like cellulosome complex. which is suggestive of extensive gene duplication as a mechanism for glycoside hydrolase diversity (M.206 Ethanol and Butanol Qi et al. albus is the only Gram-positive bacterium shown to produce type 4 fimbrial-like structures. Rincon et al. that are also present in several other R.. In silico analyses identified these polypeptides to be processive endocellulases (Cel9B and Cel48A) not previously cloned from the bacterium. these findings suggest that R. Finally.. personal communication). ScaA binds to any of seven cohesin repeats of ScaB via a specific cohesin–dockerin interaction (Ding et al. 2005). 1995. flavefaciens. 2004).

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Cellulosome organization in R. flavefaciens strain FD-1 appears similar to that of R. flavefaciens strain 17 (closely related at 97 % of 16S rRNA genes), as strongly implied by the recent discovery of FD-1 ScaA and ScaB homologues (Antonopoulos et al., 2004). The R. flavefaciens FD-1 sequencing is currently at 4 Â coverage (4.4 Mb genome) and the 1143 contigs have an average size of 3686 bp, ranging from 205 to 31 132 bp. Further analysis of the assembly revealed a cluster of genes encoding for the putative scaffoldin proteins, ScaA, ScaB and ScaC. As in strain 17, the FD-1 scaB is situated downstream of scaA, and the scaC equivalent is located upstream of scaA. Compared to R. flavefaciens 17, the architecture of the FD-1 proteins is very similar, except for the number of cohesins in ScaA and ScaB. ScaA has one less cohesin, and ScaB has two more cohesins than R. flavefaciens 17. Additionally, ScaB in R. flavefaciens FD-1 has two short prolinethreonine-rich linkers separating three cohesins, similar to linkers seen in Acetivibrio cellulolyticus. The system in R. flavefaciens therefore appears more complex than those reported previously in cellulolytic Clostridium species (Bayer et al., 2004). These comparative genomics efforts will be followed by monitoring changes in gene expression in response to exposure to different plant cell wall substrates. Indeed, a combined genomic and proteomic approach is dictated to understand the regulation and assembly of this remarkable cadre of cellulosome components, and to identify those CAZymes that have maximal degradative capacity against a given substrate. The presumptive identification of open reading frames (ORFs) from 4 Â sequence coverage of the R. flavefaciens FD-1 genome was used to develop a microarray (comprising approximately 6000 sequences). Microarray expression profiling of strain FD-1 revealed ORFs that are differentially expressed in response to growth on cellulose versus cellobiose substrates. Of the 201 cellulosomal ORFs, 57 were up-regulated and 13 were down-regulated whereas, of the 88 noncellulosomal ORFs, nine were up-regulated and 11 down-regulated. The highest levels of up-regulation were in three multimodular xylanases, which had relative expression levels of approximately 63, 50, and 25-fold above those of cellobiose-grown cells, respectively. A GH family 48 exoglucanase and a GH family 9 processive endoglucanase also had respective relative expression levels of 10- and 4.5-fold above cellobiose-grown cultures. Genes encoding the cellulosomal scaffoldin components (ScaABC) were also upregulated approximately 4.5-fold above cellobiose-grown cells. R. flavefaciens FD-1 configures the cellulosome to efficiently degrade the substrate, and constructs a more complicated cellulosome with many multimodular proteins in response to a more complex substrate. In this way, microarray analysis and the genomic information from a specialist cell wall-degrading bacterium can be used to systematically select enzymes and design an enzyme cocktail that is tailor-made for the lignocellulose substrate of choice.



The rumen possesses a natural degradative environment composed of a genomically diverse set of organisms. Often, these organisms – particularly those that are underrepresented – are missed in culture, but may supply significant metabolic contributions to other surrounding organisms in these complex environments (Ley et al., 2006; Sogin et al., 2006; Turnbaugh et al., 2006; Ley et al., 2008b; Ley et al., 2008a). Microbiologists began using cultureindependent methods, metagenomics, to circumvent low isolation numbers (less than 1 %)


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often seen with culture-based techniques in environmental samples (Handelsman, 2004; Schloss and Handelsman, 2008). Metagenomics allows genomic access to the entire population of microorganisms and allows for independent analysis of these microbes in conjunction with their natural habitat. Traditional metagenomic analyses generally begin with total extracted genomic DNA of that community. The DNA can be digested using restriction enzymes, ligated into a vector and propagated in a host, often Escherichia coli. For a sequence-driven analysis, clones can be chosen at random and subsequently sequenced. For function-driven analyses, clones can be screened for phylogenetic markers, enzymatic activity, or antibody binding. Heterologous gene expression then allows for the physiological identification of small molecules or proteins. Metagenomic studies began with traditional Sanger sequencing; however, as microscopic enumeration and colony counts were compared to the resulting numbers of microbes cultured, it became apparent that there was a large majority of organisms that were overlooked in these traditional studies (Handelsman, 2004; Schloss and Handelsman, 2007). Indeed, the sequencing and assembly of large gene insert libraries have also been hypothesized to lead to reconstruction of a near-complete microbial genome (DeLong, 2002; DeLong, 2004). As demand for genomic tools arose that would allow for a more accurate picture of the functional distribution of the microbial diversity present, the sequencing of large insert libraries (as was used traditionally in single-organism genomics) was applied to total community DNA. This allows for the screening of clones for functional diversity resulting in novel gene discovery, providing a link for genetics and functional expression for each of the selected clones. Of particular significance to this chapter, the metagenomic analysis of a bovine rumen expression identified 22 GH clones of which four potentially represent previously undescribed families of GHs (Ferrer et al., 2005). A novel polyphenol oxidase (laccase) from this bovine rumen expression library has also been identified and characterized, and it was implied that this enzyme might play a role in ryegrass lignin digestion (Beloqui et al., 2006). Massive metagenome sequencing was also recently applied to another lignocellulose-degrading community, the termite hindgut (Warnecke et al., 2007). This extensive data set showed a diversity of bacterial genes for cellulose and xylan hydrolysis, mainly from spirochete and fibrobacter species. Clearly, the termite hindgut, like the rumen, is a microbial community that is specialized towards plant lignocellulose degradation and is a potentially important source of novel enzymes for more woody substrates. With the advent of next-generation sequencing technologies, sequenced-based metagenomic approaches have strayed from cloning techniques (which introduce their own levels of bias) to a more random sequencing strategy, pyrosequencing (Ronaghi et al., 1996; Ronaghi et al., 1998; Margulies et al., 2005). Recently, this approach has been used to examine randomly sampled pyrosequence data from three fiber-adherent rumen microbiomes and one pooled rumen liquid-phase sample (Brulc et al., 2008). This genomic analysis revealed that, in the rumen microbiome, the dominant enzymes are those that attack the easily available side chains of complex plant polysaccharides and not the more recalcitrant main chains, even when cellulose is present as a substrate. Furthermore, when compared to the termite hindgut microbiome, there are fundamental differences in the GH content, with the termite hindgut microbiome containing more enzymes that are involved in the degradation of cellulose (GH5, 9, 44, and 74) and xylan (GH10 and 11). Thus, it appears that in these lignocellulose-degrading microbiomes, the CAZyme content appears to be

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diet-driven (forages and legumes or wood). Therefore, when seeking novel microbial plant cell wall-deconstructing enzymes, it is important to choose the environment that will serve as a genetic resource for plant cell wall-degrading microbial enzymes based on the substrates to be utilized.



Genomics and metagenomics are just beginning to scratch the surface of the as-yet uncharacterized CAZyme diversity present in different ecosystems. In order to fully encompass the microbial complexity and environmental impact of such diverse enzyme systems, a robust experimental approach needs to be applied utilizing a combination of sequencing technologies and microbial communities. There is optimism, however, as Overbeek et al. (2005) predicted the release of the 1000th genome in early 2008. Almost matching that figure, currently the NCBI has listed 682 complete microbial genomes, with 970 microbial genomes in progress. As sequencing technologies become more affordable and the pyrosequence read-length increases, novel enzymes are sure to be discovered. The enzyme systems discovered by these approaches will provide the starting point for characterizing and ultimately engineering, lignocellulose-degrading systems optimized for the deconstruction of biomass obtained from bioenergy feedstocks. It is anticipated that these approaches will identify thousands of novel cellulases and cellulosome components to use as a basis for the engineering and optimization of protein function. Once characterized, these proteins can be subjected to functional screenings that would link structural features with functional properties, and suggest potential mutagenesis strategies for the rational design of novel variants with enhanced degradative activity on lignocellulosic substrates. The end result will be an optimal mixture for saccharification based on empirical data and designed by substrates. Ultimately, these mixtures can be used and implemented into lignocellulosic biofuel production streams such as Consolidated Bioprocessing.

Antonopoulos, D.A., Nelson, K.E., Morrison, M., and White, B.A. (2004) Strain-specific genomic regions of Ruminococcus flavefaciens FD-1 as revealed by combinatorial random-phase genome sequencing and suppressive subtractive hybridization. Environmental Microbiology 6: 335–346. Aurilia, V., Martin, J.C., McCrae, S.I., Scott, K.P., Rincon, M.T., and Flint, H.J. (2000) Three multidomain esterases from the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17 that carry divergent dockerin sequences. Microbiology-UK 146: 1391–1397. Bayer, E.A., Setter, E., and Lamed, R. (1985) Organization and distribution of the cellulosome in Clostridium thermocellum. Journal of Bacteriology 163: 552–559. Bayer, E.A., Shoham, Y., and Lamed, R. (2000) Cellulose-decomposing bacteria and their enzyme systems, in: The Prokaryotes: An Evolving Electronic Resource for the Microbiological Community. Dworkin M., Falkow S., Rosenberg E., Schleifer K-.H., and Stackebrandt E. (eds), SpringerVerlag: New York, pp. 1–62. Bayer, E.A., Chanzy, H., Lamed, R., and Shoham, Y. (1998) Cellulose, cellulases and cellulosomes. Current Opinion in Structural Biology 8: 548–557. Bayer, E.A., Belaich, J.P., Shoham, Y., and Lamed, R. (2004) The cellulosomes: multienzyme machines for degradation of plant cell wall polysaccharides. Annual Review of Microbiology 58: 521–554.


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Beguin, P., and Aubert, J.P. (1994) The biological degradation of cellulose. FEMS Microbiology Reviews 13: 25–58. Beloqui, A., Pita, M., Polaina, J., Martinez-Arias, A., Golyshina, O.V., and Zumarraga, M. et al. (2006) Novel polyphenol oxidase mined from a metagenome expression library of bovine rumen: biochemical properties, structural analysis, and phylogenetic relationships. Journal of Biological Chemistry 281: 22933–22942. Brulc, J.M., Antonopoulos, D.A., Berg Miller, M.E., Wilson, M.K., Yannarell, A.C., and Dinsdale, E. A. et al. (2008) Gene-centric metagenomics of the fiber-adherent bovine rumen microbiome reveals forage specific glycoside hydrolases. Proceedings of the National Academy of Sciences, USA 106: 1948–1953. Davies, G., and Henrissat, B. (1995) Structures and mechanisms of glycosyl hydrolases. Structure 3: 853–859. DeLong, E.F. (2002) Microbial population genomics and ecology. Current Opinion in Microbiology 5: 520–524. DeLong, E.F. (2004) Microbial population genomics and ecology: the road ahead. Environmental Microbiology 6: 875–878. Devillard, E., Goodheart, D.B., Karnati, S.K.R., Bayer, E.A., Lamed, R., and Miron, J. et al. (2004) Ruminococcus albus 8 mutants defective in cellulose degradation are deficient in two processive endocellulases, Cel48A and Cel9B, both of which possess a novel modular architecture. Journal of Bacteriology 186: 136–145. Ding, S.Y., Rincon, M.T., Lamed, R., Martin, J.C., McCrae, S.I., and Aurilia, V. et al. (2001) Cellulosomal scaffoldin-like proteins from Ruminococcus flavefaciens. Journal of Bacteriology 183: 1945–1953. Ferrer, M., Golyshina, O.V., Chernikova, T.N., Khachane, A.N., Reyes-Duarte, D., and Santos, V.A. et al. (2005) Novel hydrolase diversity retrieved from a metagenome library of bovine rumen microflora. Environmental Microbiology 7: 1996–2010. Forsberg, C.W., Cheng, K.-J., and White, B.A. (1997) Polysaccharide degradation in the rumen and large intestine, in: Gastrointestinal Microbiology. Mackie, R.I. and White, B.A. (eds), Chapman & Hall: New York, pp. 319–379. Gal, L., Gaudin, C., Belaich, A., Pages, S., Tardif, C., and Belaich, J.P. (1997) CelG from Clostridium cellulolyticum: a multidomain endoglucanase acting efficiently on crystalline cellulose. Journal of Bacteriology 179: 6595–6601. Gerngross, U.T., Romaniec, M.P., Kobayashi, T., Huskisson, N.S., and Demain, A.L. (1993) Sequencing of a Clostridium thermocellum gene (cipA) encoding the cellulosomal SL-protein reveals an unusual degree of internal homology. Molecular Microbiology 8: 325–334. Gilbert, H.J., and Hazlewood, G.P. (1993) Bacterial cellulases and xylanases. Journal of General Microbiology 139: 187–194. Handelsman, J. (2004) Metagenomics: application of genomics to uncultured microorganisms. Microbiology and Molecular Biology Reviews 68: 669–685. Henrissat, B., and Davies, G. (1997) Structural and sequence-based classification of glycoside hydrolases. Current Opinions in Structural Biology 7: 637–644. Kakiuchi, M., Isui, A., Suzuki, K., Fujino, T., Fujino, E., and Kimura, T. et al. (1998) Cloning and DNA sequencing of the genes encoding Clostridium josui scaffolding protein CipA and cellulase CelD and identification of their gene products as major components of the cellulosome. Journal of Bacteriology 180: 4303–4308. Lamed, R., Setter, E., and Bayer, E.A. (1983) Characterization of a cellulose-binding, cellulasecontaining complex in Clostridium thermocellum. Journal of Bacteriology 156: 828–836. Leibovitz, E., and Beguin, P. (1996) A new type of cohesin domain that specifically binds the dockerin domain of the Clostridium thermocellum cellulosome-integrating protein CipA. Journal of Bacteriology 178: 3077–3084. Leibovitz, E., Ohayon, H., Gounon, P., and Beguin, P. (1997) Characterization and subcellular localization of the Clostridium thermocellum scaffoldin dockerin binding protein SdbA. Journal of Bacteriology 179: 2519–2523.

Advanced Technologies for Biomass Hydrolysis


Ley, R.E., Peterson, D.A., and Gordon, J.I. (2006) Ecological and evolutionary forces shaping microbial diversity in the human intestine. Cell 124: 837–848. Ley, R.E., Lozupone, C.A., Hamady, M., Knight, R., and Gordon, J.I. (2008a) Worlds within worlds: evolution of the vertebrate gut microbiota. Nature Reviews Microbiology 6: 776–788. Ley, R.E., Hamady, M., Lozupone, C., Turnbaugh, P.J., Ramey, R.R., and Bircher, J.S. et al. (2008b) Evolution of mammals and their gut microbes. Science 320: 1647–1651. Margulies, M., Egholm, M., Altman, W.E., Attiya, S., Bader, J.S., and Bemben, L.A. et al. (2005) Genome sequencing in microfabricated high-density picolitre reactors. Nature 437: 376–380. McCarter, J.D., and Withers, S.G. (1994) Mechanisms of enzymatic glycoside hydrolysis. Current Opinion in Structural Biology 4: 885–892. Miron, J., Yokoyama, M.T., and Lamed, R. (1989) Bacterial-cell surface-structures involved in lucerne cell-wall degradation by pure cultures of cellulolytic rumen bacteria. Applied Microbiology and Biotechnology 32: 218–222. Navarre, W.W., and Schneewind, O. (1994) Proteolytic cleavage and cell-wall anchoring at the LPXTG motif of surface-proteins in Gram-positive bacteria. Molecular Microbiology 14: 115–121. Navarre, W.W., and Schneewind, O. (1999) Surface proteins of gram-positive bacteria and mechanisms of their targeting to the cell wall envelope. Microbiology and Molecular Biology Reviews 63: 174–229. Overbeek, R., Begley, T., Butler, R.M., Choudhuri, J.V., Chuang, H.Y., and Cohoon, M. et al. (2005) The subsystems approach to genome annotation and its use in the project to annotate 1000 genomes. Nucleic Acids Research 33: 5691–5702. Pages, S., Belaich, A., Fierobe, H.P., Tardif, C., Gaudin, C., and Belaich, J.P. (1999) Sequence analysis of scaffolding protein CipC and ORFXp, a new cohesin-containing protein in Clostridium cellulolyticum: comparison of various cohesin domains and subcellular localization of ORFXp. Journal of Bacteriology 181: 1801–1810. Pages, S., Belaich, A., Belaich, J.P., Morag, E., Lamed, R., Shoham, Y., and Bayer, E.A. (1997) Species-specificity of the cohesin-dockerin interaction between Clostridium thermocellum and Clostridium cellulolyticum: prediction of specificity determinants of the dockerin domain. Proteins 29: 517–527. Pegden, R.S., Larson, M.A., Grant, R.J., and Morrison, M. (1998) Adherence of the gram-positive bacterium Ruminococcus albus to cellulose and identification of a novel form of cellulose-binding protein which belongs to the Pil family of proteins. Journal of Bacteriology 180: 5921–5927. Pohlschroder, M., Leschine, S.B., and Canale-Parola, E. (1994) Multicomplex cellulase xylanase system of Clostridium papyrosolvens C7. Journal of Bacteriology 176: 70–76. Pohlschroder, M., Canale-Parola, E., and Leschine, S.B. (1995) Ultrastructural diversity of the cellulase complexes of Clostridium papyrosolvens C7. Journal of Bacteriology 177: 6625–6629. Qi, M., Nelson, K.E., Daugherty, S.C., Nelson, W.C., Hance, I.R., Morrison, M., and Forsberg, C.W. (2005) Novel molecular features of the fibrolytic intestinal bacterium Fibrobacter intestinalis not shared with Fibrobacter succinogenes as determined by suppressive subtractive hybridization. Journal of Bacteriology 187: 3739–3751. Qi, M., Nelson, K.E., Daugherty, S.C., Nelson, W.C., Hance, I.R., Morrison, M., and Forsberg, C.W. (2008) Genomic differences between Fibrobacter succinogenes S85 and Fibrobacter intestinalis DR7, identified by suppression subtractive hybridization. Applied and Environmental Microbiology 74: 987–993. Rakotoarivonina, H., Larson, M.A., Morrison, M., Girardeau, J.P., Gaillard-Martinie, B., Forano, E., and Mosoni, P. (2005) The Ruminococcus albus pilA1-pilA2 locus: expression and putative role of two adjacent pil genes in pilus formation and bacterial adhesion to cellulose. Microbiology-SGM 151: 1291–1299. Reverbel-Leroy, C., Pages, S., Belaich, A., Belaich, J.P., and Tardif, C. (1997) The processive endocellulase CelF, a major component of the Clostridium cellulolyticum cellulosome: purification and characterization of the recombinant form. Journal of Bacteriology 179: 46–52. Rincon, M.T., McCrae, S.I., Kirby, J., Scott, K.P., and Flint, H.J. (2001) EndB, a multidomain family 44 cellulase from Ruminococcus flavefaciens 17, binds to cellulose via a novel cellulose-binding


Ethanol and Butanol

module and to another R. flavefaciens protein via a dockerin domain. Applied and Environmental Microbiology 67: 4426–4431. Rincon, M.T., Cepeljnik, T., Martin, J.C., Lamed, R., Barak, Y., Bayer, E.A., and Flint, H.J. (2005) Unconventional mode of attachment of the Ruminococcus flavefaciens cellulosome to the cell surface. Journal of Bacteriology 187: 7569–7578. Rincon, M.T., Ding, S.Y., McCrae, S.I., Martin, J.C., Aurilia, V., and Lamed, R. et al. (2003) Novel organization and divergent dockerin specificities in the cellulosome system of Ruminococcus flavefaciens. Journal of Bacteriology 185: 703–713. Rincon, M.T., Martin, J.C., Aurilia, V., McCrae, S.I., Rucklidge, G.J., and Reid, M.D. et al. (2004) ScaC, an adaptor protein carrying a novel cohesin that expands the dockerin-binding repertoire of the Ruminococcus flavefaciens 17 cellulosome. Journal of Bacteriology 186: 2576–2585. Ronaghi, M., Uhlen, M., and Nyren, P. (1998) A sequencing method based on real-time pyrophosphate. Science 281: 363. 365. Ronaghi, M., Karamohamed, S., Pettersson, B., Uhlen, M., and Nyren, P. (1996) Real-time DNA sequencing using detection of pyrophosphate release. Analytical Biochemistry 242: 84–89. Sakon, J., Irwin, D., Wilson, D.B., and Karplus, P.A. (1997) Structure and mechanism of endo/ exocellulase E4 from Thermomonospora fusca. Nature Structural Biology 4: 810–818. Schloss, P.D., and Handelsman, J. (2007) The last word: books as a statistical metaphor for microbial communities. Annual Reviews of Microbiology 61: 23–34. Schloss, P.D., and Handelsman, J. (2008) A statistical toolbox for metagenomics: assessing functional diversity in microbial communities. BMC Bioinformatics 9: 34. Schneewind, O., Fowler, A., and Faull, K.F. (1995) Structure of the cell-wall anchor of surfaceproteins in Staphylococcus aureus. Science 268: 103–106. Shoseyov, O., Takagi, M., Goldstein, M.A., and Doi, R.H. (1992) Primary sequence analysis of Clostridium cellulovorans cellulose binding protein A. Proceedings of the National Academy of Sciences, USA 89: 3483–3487. Sogin, M.L., Morrison, H.G., Huber, J.A., Mark Welch, D., Huse, S.M., and Neal, P.R. et al. (2006) Microbial diversity in the deep sea and the underexplored ‘rare biosphere’. Proceedings of the National Academy of Sciences, USA 103: 12115–12120. Ton-That, H., and Schneewind, O. (2004) Assembly of pill in Gram-positive bacteria. Trends in Microbiology 12: 228–234. Turnbaugh, P.J., Ley, R.E., Mahowald, M.A., Magrini, V., Mardis, E.R., and Gordon, J.I. (2006) An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444: 1027–1031. Warnecke, F., Luginbuhl, P., Ivanova, N., Ghassemian, M., Richardson, T.H., and Stege, J.T. et al. (2007) Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite. Nature 450: 560–565. Xu, Q., Morrison, M., Nelson, K.E., Bayer, E.A., Atamna, N., and Lamed, R. (2004) A novel family of carbohydrate-binding modules identified with Ruminococcus albus proteins. FEBS Letters 566: 11–16.

Mass Balances and Analytical Methods for Biomass Pretreatment Experiments
Bruce S. Dien



Annual ethanol production from grain in the United States is 6.5 billion gallons (BG), and is expected to grow until it reaches 10–12 BG (Westcott, 2007). At this point, it is estimated that more than 25 % of the corn harvest would be funneled into ethanol-production plants. Consequently – and given that the US annual gasoline usage is currently 140 BG – grain ethanol can only substitute up to 6.4 % of gasoline usage on an energy basis. Thus, alternative feedstocks will be needed for any continued expansion of the ethanol industry and to meet US Federal production targets for renewable fuels (36 BG per year by 2022). At present, lignocellulose is the only feedstock currently available in sufficient quantities to meet these renewable energy goals. Recently, it has been estimated that sufficient lignocellulose can be produced to replace one-third of domestic gasoline usage on an energy basis (Perlack et al., 2005). Lignocellulose can either be converted to biofuels by gasification, followed by either catalytic or biological conversion of the syngas to liquid fuels, thermal liquefaction (e.g., pyrolysis) and catalytic cracking, or by biochemical conversion to ethanol or other biofuels. Whilst many examples of each of these approaches are currently under investigation, biochemical conversion is the closest towards achieving commercialization. A good example of this is the Iogen Corporation (Ottawa, Canada), which for the past three years has been operating a pilot plant in which wheat straw is converted into ethanol. The major

Biomass to Biofuels: Strategies for Global Industries Edited by Alain Verts, Nasib Qureshi, Hans Blaschek e and Hideaki Yukawa The contribution of Dien has been written in the course of his official duties as US government employee and is classified as a US Government Work, which is in the public domain in the United States of America.


Ethanol and Butanol

steps for the biochemical process include:

. . .

Pretreatment, where the biomass is treated with a combination of physical, thermal, and chemical regimes that open up the plant cell wall structure and allow the enzymes access to the structural carbohydrates. Enzymatic saccharification, where cellulases and other hydrolytic enzymes are used to digest the pretreated biomass and extract the fermentable sugars. Fermentation, when the released sugars are fermented to ethanol or other liquid fuel products. Product recovery, which involves recovery of the ethanol, usually by distillation.

The methods discussed here apply mainly to the first three steps which, specifically, can be used to evaluate new sources of biomass, to optimize novel pretreatment technologies, to evaluate new sources of enzymes or biocatalysts, and to validate the integration of the process on an industrial basis. The analytical methods described in this chapter are akin to the tools found in a toolbox, in that they are extremely powerful when applied to appropriately designed experiments. As such, it is important to bear in mind that the objectives of engineering experiments are to supply data for process design. Process designs require data relating to mass and energy balances, unit operations, stream properties, and all energy and chemical inputs. The first step is to specify the mass balances which, for carbohydrates, are especially important in biochemical conversion processes because of their direct role in ethanol production. Few things are more frustrating than to complete an experiment and realize that the mass balances are underspecified because of one or two missed measurements. The studies cited as examples at the end of each section all include well-designed mass balances, and can thus be used to provide general guidance (see also Wyman et al., 2005).


Analysis of Feedstocks for Composition and Potential Ethanol Yield
Preparation and Storage of Samples (Dien et al., 2006; Hames et al., 2005a)

Biomass samples, if received wet, can be stabilized for long-term storage by either freezedrying or drying at a mild temperature (e.g., 50  C), preferably in a convection oven. Dried whole-plant samples can be ground through a 2 mm screen using a knife-mill (e.g., a Wiley mill). For compositional assays, the samples can be reground to pass a 1 mm screen using a cyclone mill. An inexpensive coffee bean mill and test sieves are also suitable for regrinding and size-classifying. The recommended drying temperature will not result in evaporation of all the water; as a result, samples thus prepared have 5–10 % final moisture contents. Therefore, before each experiment a small aliquot should be dried at 105  C to measure the initial moisture content. Milder drying temperatures recommended for bulk samples aim at avoiding Millard-type reactions; samples dried at 105  C are not meant to be used for further analyses. 11.2.2 Compositional Analysis

Compositional data is essential to beginning any study; typical compositional data for three maturities of switchgrass are listed in Table 11.1. Knowledge of the carbohydrate

Mass Balances and Analytical Methods for Biomass Pretreatment Experiments


Table 11.1 Results from chemical compositional analysis of different maturity switchgrass samples. All values are expressed as g kgÀ1 (dry basis) Constituent Extractables Soluble sugars Crude protein Ash Starch Cell wall fiber Arabinan Xylan Cellulose Uronic acid Klason lignin Early maturity 10 39 65 89 5 31 179 273 20 133 Mid-maturity 10 75 32 57 39 27 195 283 19 154 Late maturity 16 27 30 57 7 30 223 322 21 173

contents – that is, of soluble sugars as well as storage or structural carbohydrates – are especially important, because these values are used to calculate theoretical ethanol and sugars yields, to close carbohydrate mass balances, to determine the enzyme loadings, and to calculate the process efficiencies. The latter are typically reported as the percentage of theoretical sugars or ethanol recovered in the process. The Uppsala Dietary Fiber System (Theander et al., 1995) and NREL standard compositional laboratory analytical procedures (LAPS) (Sluiter et al., 2005a, Sluiter et al., 2005b) are standard methods for analyzing biomass matrices or lignocellulosic feedstocks (see Table 11.2). The plant composition is analyzed in a stepwise manner. (Note: NREL has generously posted their LAPs at procedure s.html.) Waxes and fats are extracted into either ether or hexane. For this, the Uppsala method suggests using a sonication bath, whereas the LAPS recommend either using a Soxhlet apparatus or a Dionex Accelerated Solvent Extractor (model 200; Dionex Corp., Sunnyvale, CA, USA). The soluble sugars are subsequently extracted in 80 % (v/v) ethanol; that is, at the ethanol concentration which is selective for monosaccharides. Water is then used to extract any water-soluble polysaccharides (e.g., fructans), followed by starch removal using amylase in acetate buffer. The final residual material is essentially pure plant cell walls. Carbohydrates present in the residual cell wall material include hemicellulose and cellulose. Commonly, both of these polymers are generically referred to as ‘structural carbohydrates,’ and are extracted after hydrolysis with H2SO4 in a two-step process (Sluiter et al., 2005b; Theander et al., 1995). The cell walls are treated with 72 % (w/w) H2SO4 at 30  C for 60 min, which causes the cellulose fibers to swell. The acid is then diluted to 4.0 % (w/v) with distilled water and the solution heated in a closed vessel at 121  C for 1 h, using an autoclave. The sugar concentrations are measured using either gas chromatography with flame ionization detection of alditol-acetate derivatives (Theander et al., 1995), with highperformance liquid chromatography (HPLC) with anion-exchange separation and pulsed amperometry (PAD) (Lee, 1996), or with HPLC using cation-exchange separation and refractometry (RI) (Sluiter et al., 2005b). If a neutral sugar analysis column is used


Ethanol and Butanol

Table 11.2 Selected compositional methods Constituent Method Principle

Below determinations are often performed stepwise Sonication in either ether or Extractablesa hexane Soluble sugarsa Starcha Structural carbohydratesa,b

Uronic acidsa,c

Acid-soluble ligninb

Klason lignina,b

Asha,b Individual assays Hemicellulose Crude proteinb Acetyl bromide lignind

Selective extraction of waxes and fats measured gravimetrically Sonication in 80 % (v/v) ethanol Selective extraction of monosaccharides measured by HPLC Extracted samples treated Released glucose measured with amylases, and sugars by HPLC or enzyme probe extracted with 80 % ethanol Sugars extracted by treating in Strong acid two-stage 72 % H2SO4 for 1 h at 30  C; hydrolysis releases cell wall then dilute to 4 % acid and associated carbohydrates hydrolyze at 121  C for 1 h in and sugars are detected by GC or HPLC closed container Liquid recovered from Hydrolyzed uronic acid two-stage hydrolysis residues detected using is treated with colorimetric assay m-hydroxydiphenyl and absorbance measured at 520 nm Liquid recovered from Hydrolyzed lignin two-stage hydrolysis components are detected is measured for UV directly by UV absorbance absorbance at 198 nm Recovered and washed residue Acid insoluble lignin is measured gravimetrically as from two-stage H2SO4 hydrolysis is dried and residue following strong acid weighed hydrolysis of cell-wall carbohydrates Above residue is combusted by Gravimetric determination following dry combustion heating at 450  C Hydrolysis with 2 M TFA for 1 h Selective acid hydrolysis of at 100  C or 121  C (see text) noncellulosic carbohydrates in non-lignified samples Total nitrogen determined by Crude protein content is equal Kjeldahl or Dumas method to 6.25 Â total nitrogen Solubilization of lignin Lignin determination based into acetyl bromide and upon standard curve; highly measurement at 280 nm correlated with Klason lignin

Sources: aTheander (1995); bSluiter et al. 2005b; cAhmed and Lavavitch (1977); dFukushima and Hatfield (2001).

for RI-HPLC analysis (such as the Shodex sugar SP0810 or Biorad Aminex HPX-87P columns), the samples must first be neutralized to pH 5–6 by the addition of calcium carbonate. In the present authors’ laboratory, sugars are analyzed routinely by RI-HPLC, using an organic acid column (Biorad Aminex HPX-87H) without neutralization, although some sugars may coelute (e.g., galactose and xylose).

Mass Balances and Analytical Methods for Biomass Pretreatment Experiments


The carbohydrate concentrations can be calculated from the following simple equation:  à sugar conc: ½mg mlÀ1 Š  solution vol: ½mlŠ Carbohydrate mg gÀ1 ¼ biomass ½gŠ  CFsugar loss  CFhydrolysis ð11:1Þ

The sugar concentration value is measured by two-stage acid hydrolysis, with the solution volume being the total liquid added through the second stage. The biomass weight is the initial weight of biomass corrected for its moisture content as measured at 105  C. (Note: as previously mentioned, the samples dried at 105  C should not be used for this assay.) The first correction factor (CF) corrects for the amount of sugars degraded during the second hydrolysis step. The extent of sugar loss is measured using pure sugar samples treated in parallel with the biomass samples. The final term corrects for the weight gained during hydrolysis. The hydrolysis of each glucan (cellulose, galactans, mannans, fructans, and starch) and pentosan (arabinan and xylan) consumes water; therefore, as shown in Equation (11.2), the monosaccharide weighs exactly 18 g molÀ1 more than the carbohydrate unit: Hexosan1 þ H2 O ! Hexose 162 g molÀ1 þ 18 g molÀ1 ¼ 180 g molÀ1 Pentosan1 þ H2 O ! Pentose 132 g molÀ1 þ 18 g molÀ1 ¼ 150 g molÀ1 ð11:2Þ

Monosaccharide concentrations can be corrected for the added water weight by dividing the sugar concentrations by 1.111 g gÀ1 (hexoses) or 1.136 g gÀ1 (pentoses). Only lignin and ash are left following complete acid extraction. These substances are recovered by pouring the acid hydrolysate through a preweighed filtering crucible or glass frit (e.g., Grouch crucible type). The washed residue is dried at 105  C, weighed, ashed at 500  C, and reweighed. Klason lignin is the weight of the recovered solids at 105  C corrected for the ash content thus determined. The NREL protocol also recommends subtracting the crude protein weight from the measured Klason lignin. Here, the crude protein content is measured beginning with a fresh sample using either the Dumas technique (combustion) (e.g., using an FP-528; Leco Corporation, St. Joseph, MI, USA) or the Kjeldahl technique (Hames et al., 2005b). Both of these methods measure total nitrogen contents; these values are in turn converted to crude protein amounts by multiplying by 6.25, an empirical coefficient that was determined based upon an average nitrogen content of 16 % in plants (Hames et al., 2005b). As a complete compositional analysis is labor-intensive, it is often more practical to measure selected components only. Soluble sugars can be extracted by sonication in water if the samples are not to be processed for determining starch contents. For total carbohydrate analysis (including soluble sugars), a two-stage acid hydrolysis procedure similar to that described above can be directly applied to native samples (Thammasouk et al., 1997). However, extraction is required when determining the lignin contents of herbaceous samples in order to avoid biasing the results (Thammasouk et al., 1997). Xylan and noncellulosic glucan can be analyzed separately from cellulose by directly hydrolyzing the biomass in a solution of 2 M trifluoroacetic acid for 1 h at either 100  C or 121  C (Albersheim et al., 1967). We have observed, however, that TFA hydrolysis of some lignified biomass samples led to lower xylan values because of incomplete hydrolysis. Samples are analyzed by mixing 0.1 g dried fiber with 2 ml of 2 M TFA in a Pyrex test tube, and sealing with a Teflon-lined screw cap. Following hydrolysis, the samples are clarified by centrifugation and directly

The fiber detergent system is an alternative method for analyzing plant cell wall composition that is commonly applied for analyzing livestock feed. 11. starch.218 Ethanol and Butanol analyzed for sugars using an HPLC system equipped with an organic acid column (Aminex HPX-87H column. 2003).5808 g gÀ1 for xylan. As fits their many roles. More recently. contracting with forage analytical laboratories may still be of value for measuring ether/hexane extractables. and avoids the need for complex analytical instruments. the difference between these values and those given for glucose and xylose arise from the weight gain associated with hydrolysis. soluble sugars. and this may be an expensive process for a given feedstock. acid detergent fiber (ADF).1 Pretreatment Pretreatment Chemistry and Equipment Plant cell walls have evolved to hold the plant upright.3 Calculating Potential Ethanol Yield The most direct use of carbohydrate data is to calculate the theoretical or maximum ethanol yield. It should be emphasized. Whilst NIR is very convenient to use. a model has been developed for determining corn stover composition before and after pretreatment (Hames et al. A typical calculation is shown for mid-maturity switchgrass in Table 11. . Inc. however.. and acid detergent lignin (ADL). Hercules. The chemical reactions used to derive the conversion factors are described in Equation (11. However. Theoretical ethanol yields are typically based upon total neutral carbohydrates. CA. 11. and crude protein. even though the maximum ethanol yield will vary depending on the biocatalyst.. xylan and cellulose contents measured in this way are inaccurate (Dien et al.. USA). and to ward against pathogens. analytical sugar standards should be run in parallel and used to correct for sugar degradation. that the lignin.2.5679 g gÀ1 for cellulose and 0. It is inexpensive to run. 2006). The xylan and cellulose contents are calculated from the NDF and ADF.3): 3Xylose ! 5Ethanol þ 5CO2 Glucose ! 2Ethanol þ 2CO2 180gmolÀ1 ¼ 2Â46gmolÀ1þ2Â44gmolÀ1 3Â150gmolÀ1 ¼5Â46gmolÀ1 þ5Â44gmolÀ1 Max:yield ¼ 92g gethanol ¼ 0:51 180g gglucose Max:yield ¼ 230g gethanol ¼ 0:51 450g gxylose ð11:3Þ The ethanol conversion factors are 0. this is unfortunate because many laboratories have been set up to perform this type of analysis in a cost-efficient manner. As with other methods that rely on acid hydrolysis.3. The animal feed industry has a long history of using near-infrared (NIR) analysis for the rapid measurement of compositional and feed properties based upon fiber determinations. Bio-Rad Laboratories.3. The results are reported as neutral detergent fiber (NDF). the building of an accurate NIR calibration requires the wet-chemistry analysis of numerous samples that span the entire expected sample space.3 11. to serve as a vascular tissue for transporting water and nutrients.

In the case of steam explosion. Mosier et al. Ethanol Yield [kg kgÀ1] Â Ethanol Sp.51 g gÀ1 for pentoses and hexoses. all of which seek the same major goal – that is. While the effects of pretreatment on the cell wall material are highly complex and still constitute an area of active research. most share similar chemical mechanisms.1 7.6477 0. Max. Alkaline pretreatments include liquid ammonia. for example.7376 0. pretreatment is an essential expense.3 Example of calculating the maximum ethanol yield for mid-maturity switchgrass Carbohydrate Concentrationa (kg tonneÀ1) Soluble sugars Glucose Fructose Sucrose Storage CHO Fructan Starch Structural CHO Arabinan Cellulose Galactan Mannan Xylan Total a b Conversionb factor (l kgÀ1) 0. Jørgensen et al.0 l tonneÀ1 ¼ 2. There are numerous pretreatments. 2005. and 0.9 204. Among the numerous pretreatment methods that have been described (Dien et al. to open up the cell wall matrix and allow the cellulase access to the individual cellulose fibers.789 kg lÀ1. As conversion rates for native plant cell wall material treated solely with commercial cellulases are typically lower than 20 %.1 19. Mosier et al. and lime. the explosive release of pressure also serves to reduce the average particle size.0 28. c 10. partial hydrolysis of the lignin.Mass Balances and Analytical Methods for Biomass Pretreatment Experiments 219 Table 11. and to swell or de-crystallize the cellulose fibers (Dien et al. Ammonia Fiber Expansion (formerly explosion.. Conversion factor: Max.7212 0.. rely on acid hydrolysis of the xylan. 2005a..2 2. [l kgÀ1].567 g gÀ1 for hexosans.8 456. to melt the lignin polymers. specific volume of ethanol (25  C) ¼ 0. which also often includes a mineral acid catalyst.. Liquid hot-water also relies on a partial acid hydrolysis of carbohydrates. These pretreatments rely on alkali to dissolve the xylan and (in part) the lignin. Jørgensen et al. albeit at a less acidic pH.6477 0.7212 0. 2007). plant cell walls are highly resistant to enzymatic digestion. to displace lignin.7376 Contributionc (l tonneÀ1) 9. All of these also rely on high reaction temperatures (160–200  C þ ) to enhance the rate of acid hydrolysis. lime is often .7212 0.8 0.7212 0.5 14 10 51 0 39 27 283 10 4 195 On a dry basis.580 g gÀ1 for pentosans.. 0. Ethanol Yield ¼ 0. and acid swelling of the cellulose fibers.7212 0.5 34. Liquid dilute-acid and steam explosion.1 6. AFEX).40 US gallons tonneÀ1. Each of these alkaline-based treatments has unique features.6833 0.9 143.. effective pretreatments are known to remove xylan. Alkali also disrupts hydrogen bonding among the cellulose fibers. and to achieve thermal disruption of the highly ordered cellulose structure. Vol. 2005a. 2007). 2005.

(1996). Several research groups (e. Routinely. while the vessel diameters should be kept to a minimum to ensure uniform heating. AFEX is applied at very high pressures and relies upon an explosive decompression to further disrupt the plant cell walls. Klason lignin. (1998) and Palmqvist et al. Lloyd and Wyman.3. The major disadvantage of this technique is the absence of mixing. On the other hand. mild NH4OH. care must be exercised when filling the reactors so as to leave a sufficient headspace to allow for the thermal expansion of water when the mixture is heated to the reaction temperature. solid loadings are typically limited to 10–15 % (w/w). A subsample is further dried at 105  C to measure the absolute moisture content of the partially dried cake.. Notwithstanding this . 1998). 2003) react biomass in customfabricated stainless steel pipe reactors that are heated by immersion in a fluidized heating bath. the methods for further optimizing the reaction conditions and evaluating the sugar and ethanol yields remain the same.g. sugars. Next..... USA) or by centrifugation (Eddy et al. and lime) can be carried out in simple glass containers because they are performed at atmospheric pressures. USA). Available assays to monitor the reaction conditions can be subdivided into chemical. and include the determination of the contents: solids. The major advantages of this technique are its reasonable costs. Lloyd and Wyman (2005) discussed heating a stirred 1 liter Parr reactor in a fluidized sand bath as a means of achieving faster heating times. or by pressing through a finely pored nylon cloth (e. Recently. the amount of suspended solids in the liquid cannot be directly measured because the acid or base becomes more concentrated as the water evaporates (Ehrman and Himmel. which include sugar degradation products. and fermentation assays (see Table 11. it was observed that metal ions leaching from a metal-surfaced reactor could slightly enhance the rate of furfural formation (Kumar and Wyman. Moline.4. In contrast. 1994). PA. microwave-heated reactors equipped with automated temperature control and high-pressure stirred reactors have also proved to be useful (e.220 Ethanol and Butanol applied at mild temperatures (albeit over long time periods). no standard laboratory equipment has yet been manufactured for modeling industrial biomass pretreatment. Dilute-acid and liquid hot-water or ammonia pretreatments are typically carried out at 160–200  C in pressurized vessels. and Parr Instrument Company. enzymatic. Autoclave Engineers. 11. and the ability to run multiple reactors in parallel. Upon pretreatment. Whichever pretreatment(s) is (are) applied. The recovery is incomplete since some liquid is retained within the solids. chemical assays are applied in combination with either enzymatic or fermentation assays.4). and inhibitory chemicals. IL.g. the short heating and cooling times (by quenching in a water bath). consequently. the liquid portion is recovered by vacuum filtration through thick glass fiber filters. Nonetheless.g. Moreover. custom-fabricated steam explosion reactors have been described by Nguyen et al. Mild alkaline pretreatments (e.g. 2008). Miracloth. Calbiochem. Finally. Unfortunately. San Diego. It should be noted that when a nonvolatile acid or base is used as a catalyst for pretreatment. alkaline hydrogen peroxide. the solids are extensively washed with distilled water and the washed cake dried at 50  C. CA. Erie.2 Chemical Analysis of Pretreated Biomass Methods for the chemical analysis of pretreated samples are summarized in Table 11.

2006. cerevisiae at 35  C for 72–144 h..g.g.. and McMillan. 1998.. cSluiter et al. 2006.. dDien et al. 1996. 2004. Ethanol can be measured by enzyme assay. 2005b. HPLC.. solids should not be dried prior to assay. 2006. solids should not be dried prior to assay Ethanol produced following cotreatment with cellulase (15 FPU gÀ1 glucan) and fermentation with S.. fDowe ´pez et al. eBrown and Torget.Mass Balances and Analytical Methods for Biomass Pretreatment Experiments Table 11.4 Selected analysis of pretreated biomass Fraction Liquid hydrolysate Solidsa Monosaccharidesb Soluble carbohydratesb Assay Gravimetric determination of solids from filtrate or supernatant after drying at 105 C Sugar concentration measured by HPLC Sugar concentration measured by HPLC following acid hydrolysis for 1 h by 2 M TFA (100  C) or 4 % (v/v) H2SO4 (121  C) Furfural and HMF concentrations and other inhibitors measured by HPLC Gravimetric determination of solids after drying at 105  C Released sugars measured by HPLC following two-stage H2SO4 hydrolysis of dried (e.. bSluiter et al. <55  C) solids Released glucose measured by HPLC following treatment of solids with cellulase (60 FPU gÀ1 glucan) for 72–144 h. hChen et al. or GC Enzymatic hydrolysis of neutralized pretreated biomass – methods vary Ethanol fermentation using microbe that ferments sugar mixtures – methods vary Result 221 % biomass partitioned to liquid phase – not appropriate for many pretreatments Concentration of readily fermentable sugars Sum with monosaccharides to give % of carbohydrates extracted Sugar degradation productsb.g and inhibitorsh Water-washed solids Solidsa Cellulose and hemicellulosec Hemicellulosed % Biomass that remains intact and needed mass balance information Concentration of cellulose for enzyme loading % Xylan not extracted and concentration of xylan for enzyme loading % Available glucans and xylose for possible fermentation Cellulase digestibilitye SSFf % Ethanol realized based on cellulose content Whole hydrolysate Enzymatic digestibility SSF Yield of sugars for fermentation by SHF Overall ethanol yield from biomass Sources: aEddy et al. 2001.. <55  C) solids Released sugars measured by HPLC following TFA hydrolysis of dried (e. gLo .

In the liquid hydrolysate (LH) phase. this product is measured directly by HPLC. the LH can be determined by measuring the density of the recovered hydrolysate liquid (Density) and knowledge of the initial amount of Biomass (g. and lignin balances. Finally. When the syrup is dilute enough and the pretreatment reactor is closed (i.e. it should be noted that the glucose balance (Equation (11. The latter technique is preferred as it is more sensitive and has shorter analysis times.. Insoluble Solids (g. glucose molecules are present in both the Liquid and Solids phases of the hydrolysate. the suspended and soluble solids significantly expand the end volume. the suspended and soluble solids can be determined from the starting amounts of biomass and recovered washed cake. 2006).4)) could also have been presented using units of moles and the appropriate stoichiometric coefficients. all on a dry basis.4) should be based upon dry weight as determined at 105  C. either by ion-moderated partition chromatography or by reverse phase using a C18 column and UV detection (l ¼ 277 nm).. when the solution is more concentrated. whereas glucans are measured as glucose by HPLC following complete acid hydrolysis of the liquid either in 4 % H2SO4 or 2 M TFA. The glucose balance is represented by Equation (11. the glucose can exist either as a monosaccharide or as soluble glucans. dry basis). this volume cannot be measured directly because a significant part of the syrup is retained in the filtration cake. It is this latter technique that is generally applied. In addition to solids. Following pretreatment. the liquid volume is the sum of the initial moisture content of the biomass sample and of all liquid volumes that were added during the course of the experiment. The amounts of Solids [g] and Biomass [g] used in Equation (11. As noted above. dry basis). the more important balances used to describe pretreatment processes are the glucose. The amount of glucose that comigrates with the solids is measured by analyzing the washed solids for cellulose content and correcting for hydrolysis. Since hydroxymethylfurfural (HMF) is the predominant product of glucose degradation. Glucose amounts are directly measured by HPLC. and total amount of added water (Water): Liquid ½lŠ ¼ Water ½gŠ þ Biomass ½gŠÀInsoluble Solids ½gŠ Density ½g lÀ1 Š ð11:4bÞ . Another quantity that is important to determine is the volume of liquid hydrolysate (LH).4): Glucosein ½gŠ ¼ ðGlucose½%Š þ 0:653 Sucrose ½%Š þ 1:111  Starch ½%Š þ 1:111 Cellulose ½%ŠÞ  Biomass ½gŠ Glucoseout ½gŠ ¼ ðGlucose ½g lÀ1 Š þ 1:111 Glucan½g lÀ1 Š þ 1:429 HMF½g lÀ1 ŠÞ  Liquid ½lŠ þ 1:111Cellulose ½%Š  Solids ½gŠ ð11:4Þ The glucose amount present in the sample can be readily calculated from the compositional data and from the biomass amount initially added on a dry basis. when there are no evaporative losses). since fructose decomposes into HMF more readily than does glucose (Dien et al. where the LH is diluted equally with 4 M TFA and heated in a sealed glass test tube for 1 h at 100  C. The compositions are listed as weight % of carbohydrate per weight of biomass or solids. Notably. xylose. In this case. However.222 Ethanol and Butanol technical hurdle. a fructose balance is needed for samples rich in fructose and sucrose.

62 % of the lignin was removed while 85 % and 100 % of the xylan and glucan were retained. 2007). respectively. Listed runs were ordered according to their combined severity factor (CSF). temperature (165–183  C) and sulfuric acid loading (0. as follows: Xylosein ½gŠ ¼ 1:364 Xylan ½%Š  Biomass ½gŠ Xyloseout ½gŠ ¼ ðXylose ½g lÀ1 Š þ 1:364 Xylan ½g lÀ1 Š þ 1:562 Furfural ½g lÀ1 ŠÞ ð11:5Þ Liquid ½lŠ þ 1:364 Xylan ½%Š  Solids ½gŠ In this case. dissolved xylan. It was determined that a greater lignin removal coincided with an increased enzymatic digestibility and. the xylose pool originates solely from xylan and is lost by decomposition into furfural. 2003).6–1.6):  ! Temp ½ CŠ-100 -pH CSF ¼ log10 time½minŠ  exp 14:75 ð11:6Þ where ‘time’ and ‘Temp’ represent the reaction time and temperature. Pretreated washed solids were each analyzed for dry weight. intact xylan.5–1. and furfural – mass closures averaged 93. Following pretreatment. The methods for analysis are similar to those described for glucose and the various parameters of this equation. which is defined as described in Equation (11. it is still useful to analyze the solids for Klason lignin content and to calculate the percentage of lignin that is removed. as this constitutes an important parameter when considering some pretreatments (Kim and Lee..7. However. a complete xylan mass balance was described that included as inputs the following variables: xylose. can be co-measured with those of the glucose balance equation.7 Æ 2. Chemical analysis was also used for optimizing the steam explosion conditions for the pretreatment of corn stover (Schell et al. The objective of this specific pretreatment was to optimize the steeping conditions (time. The chemical complexity of lignin does not readily lend itself to a similar mass balance modeling. The optimal xylose yield was found to be 70 % and at a CSF of 1. 11. and percentage solids) for ensuring the maximum removal of lignin and maximal retention of carbohydrates.6 %. 2007). The pH values are typically measured from the hydrolysate following pretreatment at room temperature.3. and glucan contents.41 %). respectively.Mass Balances and Analytical Methods for Biomass Pretreatment Experiments 223 A balance equation similar to that of glucose can be formulated for xylose.. The reaction conditions were varied for time (3–12 min). Corn stover was treated in a continuousflow steam explosion reactor with a capacity of 32 kg (dry basis) hÀ1. The CSF is an empirical function that greatly simplifies the experimental design for dilute-acid pretreatments because it allows for three parameters to be optimized simultaneously. ammonia hydroxide concentration. at optimal conditions [15 % (w/w) NH3 solution at 60  C with a 12 h soak time]. xylan. lignin.3 Case Studies The chemical analysis of biomass following pretreatment was used extensively to guide the development of ammonia steeping as a corn stover pretreatment (Kim et al. .

and to run reactions with various cellulase loadings. Digestion is most often performed using the washed wet cake. The cellulose concentration is purposely kept low. and for this case xylose can be measured directly following pretreatment. then the mass balance equation is . independent of enzymes. The cellulose content should be known from prior chemical (or NIR) analysis. Microbial contamination is prevented by adding an antimicrobial agent such as thymol or sodium azide (Dien et al. the value of this parameter can be estimated as the difference between the cellulose content initially present in the biomass sample and the complete glucan content of the LH. provided that the xylose concentration is simultaneously monitored with glucose by HPLC. Alternatively. Substituting whole hydrolysates for washed cakes would be of particular interest for process designs that do not comprise units for performing liquid–solid separations.224 Ethanol and Butanol 11. as a means of obtaining conversion rate data. although it is often beneficial to supplement these activities using a commercial xylanase preparation. Enzymatic digestion results can be used to extend as needed the mass balance equation described earlier. as described previously. In these cases. when completely hydrolyzing xylan into xylose. the solids are not dried prior to the digestion assay. 2001). because the cellulase enzymes are inhibited by relatively low concentrations of released glucose via a phenomenon termed ‘product inhibition. Most often. if all of the pretreated hydrolysate is successfully transferred from the pretreatment reactor to the digestion flask. Instead.8–5.’ The digestion reaction can be described as follows:   ðFinal Glu½glÀ1 Š-Enzyme Glu½glÀ1 ŠÞ Rxn Vol½lŠ  100 Efficiency of Glu Release ½%Š ¼ 1:111  Cellulose½%ŠÂ Solids½gŠ ð11:7Þ The final glucose concentration (Final Glu) is measured directly using HPLC. Specifically. as even gentle drying at 50  C could collapse the cell wall structure and thus impede digestion (Dowe and McMillan.0) to a cellulose concentration of approximately 10 g lÀ1 and subsequently digested at 50  C for 100–144 h with 60 filter paper units of enzyme per gram of cellulose (Brown and Torget. the release of xylose is aided by the typical presence of xylanase activities in commercial cellulases.. the wet solids are diluted in a 50 mM sodium citrate buffer (pH 4.4 Enzymatic Extraction of Sugars Pretreatment conditions are most commonly optimized to maximize the enzymatic conversion of cellulose to glucose using commercial cellulase blends. the xylose release can be followed during the enzymatic reaction.7) by Enzyme Glu [g lÀ1]). it is important to ensure that the solids have a uniform moisture content of known value. it is important to subtract their contribution to the glucose pool (represented in Equation (11. This approach offers several advantages. It is also useful periodically to sample the digestion reactions. Enzymatic saccharification can also be carried out using whole hydrolysates. In addition. In a similar manner to glucose. for other types of pretreatment. 1996). 2008). Dilute acid pretreatments are often effective. The amount of Solids is calculated on a dry basis relative to the samples treated at 105  C. Alternatively. an accurate estimation of the cellulose content is needed for determining the optimal cellulase enzyme loading. the efficiency of xylose release can be also calculated. First. The results from these types of experiment can be used to further differentiate and tailor design pretreatment conditions to specific biomass sources. As most commercial enzyme blends contain residual sugars.

Nevertheless. Commercial suppliers of cellulolytic enzymes include: Genencor (Palo Alto. and Rohm-AB Enzymes (Rajamaki. many pretreatments do not completely hydrolyze the xylan and. the glucose concentration [g lÀ1] of the diluted solution is measured by HPLC. Treating the whole hydrolysate provides an opportunity for the enzymes to saccharify these soluble carbohydrates as well as the insoluble ones. (Jupiter.. Critical aspects for using this method are that glucose is sufficiently concentrated in the hydrolysate to be accurately measured by HPLC following dilution. For this reason. and the whole hydrolysate sample is added to the water before bringing the dilution up to its final targeted volume.g. a representative sample of the whole hydrolysate is withdrawn. Commercial cellulases are a complex mixture of enzymes that vary considerably in their cellulase contents and other undisclosed activities (Nieves et al. (Cambridge. For this. this approach is not without its disadvantages. The reader is directed to Hodge et al.4)b). Inc.. Finland).g. 10  dilution). as a result. 2007). Dyadic International.Mass Balances and Analytical Methods for Biomass Pretreatment Experiments 225 dramatically simplified as all of the glucose released can be directly related to the biomass initially present in the sample. It should be noted that Equation (11. Kabel et al. An important consideration for these assays is the sourcing of the enzymes used... MA). Canada). Iogen (Ottawa. Denmark). 2006. it is good practice to evaluate multiple cellulase formulations. In this case. Genencor and Novozymes recently completed . Occasionally. and the glucose content (Final Glu) of the whole hydrolysate is measured as follows:    à Whole hydrolysate ½gŠ Final Glu ½gŠ ¼ GlucoseHPLC g lÀ1  Final volume ½lŠ  Sample ½gŠ ð11:7bÞ The yield efficiency can now be calculated using the final glucose content and correcting for any glucose inadvertently added with the enzyme preparations (Enzyme Glu [g]):   ðFinal Glu ½gŠ-Enzyme Glu ½gŠÞ  100 ð11:7cÞ Efficiency of Glu Release ½%Š ¼ 1:111  Cellulose ½%Š  Solids ½gŠ where Solids [g] is the total amount of pretreated biomass present in the whole hydrolysate on a dry basis. there is a simpler protocol available. 100 g lÀ1 þ sugars). CA). Notably. notably that whole hydrolysates almost always produce poorer glucose yields than washed solids (Merino and Cherry. 2008). Whilst one way to correct for this is to calculate the actual volume of the liquid hydrolysate as previously shown (Equation (11. a Sample [g] of the Whole Hydrolysate [g] is diluted in distilled water sufficiently to minimize the effect of insoluble solids on the final volume (e. 1998. neither can the results be compared directly with previously published data. Novozymes A/S (Bagsvaerd. the volume expansion associated with suspended and dissolved solids must be accounted for.7c) is on a mass basis. 1996). Verenium Corp.. Regardless of the commercial source. it may be desirable to carry out enzymatic digestions on undiluted whole hydrolysates to obtain more concentrated sugar streams (e. cellulase blends should be supplemented with b-glucosidase to a final activity of 40–64 units gÀ1 cellulose (Brown and Torget. FL). or the calculated glucose yield efficiency will be lower than the actual value. Second. Dien et al. (2009) for a further discussion of treating high-solids for the special case of washed pretreated biomass cake treated enzymatically at high-solids. the xylan pool is converted into pools of various oligomers that cannot be fermented by most ethanol-producing microbes.

NJ. Pretreated biomass can be converted to ethanol using two .4. 11. this was achieved by using a magnetic stirrer while aliquots were transferred with a 5 ml pipetter. Moreover. Adney and Baker (1996) present a detailed protocol for measuring filter paper units.9–93. the pretreated material was diluted in a medium composed of distilled water. The liquid fraction was analyzed for total xylose (monomeric and oligomers) and glucose yields.50.8). Inc. the scintillation vial caps were retightened after the vials equilibrated at 50  C. The transfer of equal amounts of solids was confirmed by drying a test set and comparing the total solids transferred to each sample.. Lastly. especially those attained from cellulose. Pierce Biotechnology. employing tips with their ends clipped off as a simple means to prevent blockage from solid particles. 2001). Genencor has released its newly developed cellulase under the trade name AcceleraseÔ 1500. 2007). this preparation is reported to have an enhanced b-glucosidase activity. The washed solids were treated at 1 % (w/w) solids with a cellulase loading of 60 FPU gÀ1 of cellulose. are useful indicators to directly guide pretreatment development. the pretreated material was subsequently dispensed into disposable scintillation vials. It was also determined that total yields of 90 % could be maintained even when the cellulase loading was decreased to 6 FPU gÀ1 cellulose. 11. xylanase and a surfactant can improve yields attained with various sources of biomass (Murnen et al. Dien et al. (2008) described a convenient assay for optimizing enzyme loadings for a common pretreated substrate. The homogeneity of the solutions was kept optimal by placing the vials on a tube roller (Bellco Glass. An example of screening commercial cellulase blends for their full range of enzymatic activities can be found in Dien et al.226 Ethanol and Butanol the development of new cellulase blends as part of a US Department of Energy (DOE) sponsored research project. digested at 50  C for 72 h.1 Case Studies Lloyd and Wyman (2005) used a combination of chemical and enzymatic analysis to determine the optimal conditions for pretreating corn stover with dilute acid. Perhaps the most unique aspect of these studies was that the pretreatment conditions were optimized based upon total glucose and xylose recovered following enzymatic saccharification. (2008). and the optimal Log10CS was approximately 1. and analyzed for glucose and xylose. IL. Finally. It is also convenient to measure the crude protein concentration for enzyme blends using the bicinchoninic acid protein assay (BCA Protein Assay. USA) for enabling the comparison of different formulations. In this case. and thymol.0 % of maximum.5 Fermentation of Pretreated Hydrolysates to Ethanol Ethanol yields.. Various pretreatment conditions were compared based upon a total yield that included glucose and xylose released in the two stages. Various enzyme combinations were added to the hydrolysate and digested at 50  C for 72 h. USA). sodium citrate buffer (pH 4. Moreover. Rockford. Yields were 78. care was taken to ensure that the whole hydrolysate was kept well mixed when being transferred to the vials. empirically adding pectinase. Vineland. Cellulase blends should be stored at 4  C and their activities checked every six months (Dowe and McMillan.

corn stover was treated with dilute-acid at various pHs. it is essential that homogeneous samples be taken so that all concentrations remain constant throughout the fermentation. the culture ferments these to ethanol. galactans. mannans. fructans. as they can all be fermented by Saccharomyces. and residence times. cellulose. and 25 (FPU gÀ1 cellulose) at 10 % (w/w) solids. For this. then. glucose. while pentosans would include arabinan and xylan. while pentoses would include xylose and arabinose. avoids the presence of large amounts of solids in the bioreactor. for Saccharomyces yeast fermenting washed solids. In SSF experiments. fructose. Hexoses would include glucose and fructose. As a result. on the other hand.dependent. Hexosans would include cellulose. it goes without saying that if the fermentation is sampled at multiple timepoints. Moreover. Calculating the maximum ethanol yield is also organism. (2003) used SSFs to evaluate the dilute-acid steam explosion of corn stover. sucrose.Mass Balances and Analytical Methods for Biomass Pretreatment Experiments 227 general schemes: simultaneous saccharification and fermentation (SSF).1 Case Studies Tucker et al. and is calculated from the mass amount of monosaccharide equivalents added to the fermentation mix.and process. The solids were recovered from each run. only cellulose would be included in Equation (118b). SSF requires lesser amounts of enzyme because it circumvents end-product inhibition and avoids opportunities for microbial contamination. for Saccharomyces fermenting whole hydrolysates. Usually. starch. as the hydrolytic enzymes free the glucose and possibly xylose molecules. galactans. with the conditions being aggregated by using the combined severity factor as defined previously. the cellulase and fermenting microbe are added simultaneously to the hydrolysate. as this constitutes an added safeguard for deriving an accurate final ethanol yield. 11. The monosaccharide equivalents can be calculated as shown by the equation below. and separate hydrolysis and fermentation (SHF). the input volume is the total liquid volume at the beginning of the fermentation. washed extensively with distilled water. The cultures were . In contrast.5.8):   Ethanol ½g lÀ1 Š  Volume ½lŠ  100 ð11:8aÞ Process Eff ½%Š ¼ 0:51  MonoSacc ½gŠ MonoSacc ½gŠ ¼ Hexoses ½gŠ þ Pentoses ½gŠ þ 1:053 Sucrose ½gŠ þ 1:111 ÂHexosans ½gŠ þ 1:136 Pentosans ½gŠ ð11:8bÞ The lower term gives the maximum amount of ethanol that can be expected. the expanded volume because of ethanol production may need to be included in this calculation. if a xylose-fermenting microorganism were to be used. Also. and mannans would be included. temperatures. the end result can be summarized by calculating the process efficiency (Equation 11. and so on. and fermented with Saccharomyces cerevisiae D5A in the presence of Iogen cellulase at 5. in SHF experiments the biomass is saccharified with cellulases prior to fermentation. and also allows for the independent control of temperature and pH for saccharification and fermentation. However. Additional fermentation flasks can be included that are sampled only at the end of the experiment. Regardless of which process is favored. 15. SHF. In the case where fermentations are conducted using more concentrated media. then xylan would also need to be included.

2008). SSF data can be used to evaluate and screen different sources of biomass for their conversion potential. One key difference between these types of study and the more classical approaches outlined above is that. Therefore. For example.. it is essential that ethanol yields are correlated to the amount of biomass initially present in the sample treated. In this experiment. One commonality. and (ii) the results are expected to be more meaningful as they reflect actual ethanol production. is that very careful attention must be paid to mass balances when measuring success. the fermentation efficiencies reported were based solely on the cellulose contents. 11 samples of bunch grasses were thus compared for ethanol yields using an autoclave dilute-acid pretreatment (Anderson et al. Notably. strain D5A is not a recombinant yeast and as such it does not ferment pentoses. and the yields calculated as percentages of the theoretical ethanol production. . The assay was designed so that the same bottle was used for pretreatment and fermentation. the lower-severity treatment applied leads to a much lower final ethanol yield. One consequence of applying the assay to the washed cake is an improved fermentation. because these translate into higher ethanol concentrations. The ethanol yields ranged from 72–92 %. Cellulose contents were measured for each sample of washed cake. which in turn removes the need of any form of conditioning prior to fermentation. cerevisiae D5A in the presence of standard concentrations of cellulase (5 FPU gÀ1 biomass) for 72 h. but also other soluble components such as xylose or aromatic compounds that interfere with the enzymatic hydrolysis. as this makes it suitable for assaying larger sample sets and also greatly simplifies the mass balance calculations. As engineered microbes for fermenting mixed sugars become more widely available with improved ethanol productivities.. other than neutralization. this technique is only suitable for ranking sets of biomass samples respectively to their intrinsic ease of conversion. The whole hydrolysates were neutralized with Ca(OH)2 and fermented with S. There are two important advantages of SSF over enzyme digestion assays in determining potential ethanol yields: (i) lower enzyme loadings are required due to the intrinsic design of the SSF process. 11. the investigators are expanding their research to include a fully integrated process approach to the evaluation of pretreatments. This type of process also tends to favor the use of higher solid concentrations. Nevertheless. Consequently. the pretreatment is directly optimized for product yield and productivity. and not for predicting expected ethanol yields in a commercial process.228 Ethanol and Butanol periodically sampled over 180 h. cellulose digestibility may be partially sacrificed to minimize the formation of furfural from xylose. in this respect. however. by including a fermentation step.6 Feedstock and Process Integration It is widely expected that the achievement of an efficient fermentation of glucose and xylose will be required to ensure the commercial success of cellulosic ethanol. this variable was in turn used to calculate enzyme loadings and fermentation efficiencies. On the other hand. the lower-severity pretreatment temperature that was used has the advantage of lowering inhibitor production. Consequently. Washing removes not only any soluble inhibitors that could lower the ethanol yield or stall the fermentation. In addition. the grass samples were treated with dilute sulfuric acid by heating at 121  C for 1 h.

and the need to adapt protocols to the use of experimental microorganisms capable of fermenting sugar mixtures. Clearly. at higher solid concentrations. and this will most likely be the modus operandi that will be favored by commercial plants. Furthermore. and (iii) the impact of novel equipment designs for agitating heavy slurries. Operating at higher solid concentrations will undoubtedly place greater demands on enzyme formulations and fermentation microbes. and detailed protocols describing these techniques are readily available. Today. which is 88 % of the maximum possible yield. the trend in pretreatment studies is towards combined pretreatment and fermentation. Moreover. Fermentation studies are more complex because of the added protocol steps. after which the recovered whole hydrolysate was mixed with cellulase (11 FPU gÀ1 cellulose) and fermented using xylose-fermenting Saccharomyces 424A (LNH-ST). (ii) the influence of changes in xylan solubility. further research is required to adapt current methods to operate at high solid concentrations. further conditioning is required to remove any byproducts of the pretreatment that might inhibit fermentation. 2007). more complex mass balances are required that can account for volume changes from dissolved and soluble solids. corn stover was pretreated at 190  C for 15 min and 16 % (w/ w) solids.7 Perspective Robust methods have been developed for analyzing the results from biochemical conversion experiments. the practitioner in this field should promote the production of laboratory data that are more relevant to the evolving parameters when implemented at the commercial scale. (2005b) investigated the liquid hot-water pretreatment of corn stover for conversion to ethanol. Gregory Kennedy. Most often. Newly available enzymes suitable for SHF will no doubt resolve some of these concerns. and Patricia O’Byran for their critical readings of the manuscript. The SSF was completed within 60 h and the final ethanol concentration was approximately 22 g lÀ1. thus adding complexity to the mass balance. One point important to consider here is that the liquid hot-water pretreated hydrolysate was directly fermentable by the yeast.1 Case Study Mosier et al.6.Mass Balances and Analytical Methods for Biomass Pretreatment Experiments 229 11. operating at higher solid concentrations also offers opportunities for studying new phenomena. Despite these advances. Acknowledgments The author thanks Michael Cotta. 11. the increased concern regarding byproducts of pretreatment that may partially or completely inhibit fermentations. including: (i) the influence of changes in viscosity and water activity on yields. as it is common to observe both rapid declines in sugar yields and stalled fermentations when solid concentrations are used rather than dilute solutions (Merino and Cherry. Here. .

D. Biotechnol. J. Ximenes.H. and J. 54–61 (2006). M. A simplified method for accurate determination of cell wall uronide content. Soluble and insoluble solids contributions to high-solids enzymatic hydrolysis of lignocellulose..D.C. 13–21 (2008). H. Kumar and C. Laboratory Analytical Procedure. Ahmed and J. Chromatogr. Sluiter. NREL/TP-510-42625 (2005b).B. Biochem. Sarath. Boca Raton. Weimer. Lamb. Klip. Techn. Balan. Res. 1104. Pretreatment of corn stover by soaking in aqueous ammonia at moderate temperatures.D. Brown and R. Laboratory Analytical Procedure. P. J.E. R. 145. 8940–8948 (2008). Appl. Baker.. Eddy. 105–108. Brandon and J.J. Hames. Chen.A.S.S. Dien. McMillan. Extraction and isolation of lignin for utilization as a standard to determine lignin concentration using the acetyl bromide spectrophotometer method.I.F. C. Hodge. Peterson. Lee. P. Himmel.230 Ethanol and Butanol References B. (2008).A. Laboratory Analytical Method. Laboratory Analytical Procedure. 81–92 (2007). Chambliss. Biofuels. 880–891 (2006). A.Preparation of samples for compositional analysis. Laboratory Analytical Procedure. C. Sluiter. B. 361–365 (1977). Enzymatic conversion of lignocellulose into fermentable sugars: challenges and opportunities.. Y. NREL/TP-510-42628 (1996). 57–63 (2006). in Handbook of Industrial Biocatalysis. 5216–5225 (2008). J. Skory.D. K. Karim. Technol.J.F. 99 (18). Karr. R. Nevins. 340–345 (1967).L. Thomas.V. A.J.P. Biochem.A.K. Scarlata. A method for the analysis of sugars in plant cell-wall polysaccharides by gas-liquid chromatography. 49. A. Moniruzzaman.E. C. Carbohydr. S. Biomass Bioenergy. Hames. Mowery. Food Chem. J. R. Roberson. lignocellulosic biomass hydrolysis and fermentation. Biotechnol. Walsum and C. B. Technol. Iten and C.R. NREL-018 (1998). C. V.A. 30.). Chemical composition and response to dilute-acid pretreatment and enzymatic saccharification of alfalfa. Scarlata and A. J. Okafor and C. 136–140. Measurement of cellulases activities. Enzymatic saccharification of lignocellulosic biomass. Converting herbaceous energy crops to bioethanol: a review with emphasiss on pretreatment processes. The impact of dilute sulfuric acid on the selectivity of xylooligomer depolymerization to monomers. E. Rapid biomass analysis: new tools for compositional analysis of corn stover feedstocks and process intermediates from ethanol production.B.S. W. NREL/TP-510-42630 (2001). Sluiter and D.J.A. Templeton. Dien. Dowe and J. Vogel. Iten.D. O’Bryan. A.Y. S. L. M.W. NREL #009 (1996). van der Maarel. 720. Kabel. Simultaneous saccharification and fermentation of pretreated biomass: improving mass balance closure. Roth and D. 93. Biorefin. B. Biores. reed canarygrass. G. High-performance liquid chromatography method for simultaneous determination of aliphatic acid. Hatfield. Anderson. McMillan. M. Sluiter. Kim and Y. V. 99. P. 119–135 (2007). Dien. D. English and A. and switchgrass. Ho (ed. SSF experimental protocols.M. F.J. Albersheim. R. Kristensen and C.P. B. S. C. 1. Chromatogr. Torget. Ehrman and M. Schell. Res.A. Hames. Biotechnol.. Cotta. B. Food Biochem. Taylor and Francis Group.F. P. Lavavitch.J. X. J. NREL/TP-510-42620 (2005a). . M.E..S. Carbohydrate analyses with high-performance anion-exchange chromatography. D. Enzymatic extraction of sugars from AFEX and liquid hot-water pretreated distillers’ grains and their conversion to ethanol. Lee. R. 3133–3139 (2001). Laboratory Analytical Procedure. aromatic acid.C.R.B. 343. FL. 99–104 (1994).. Castleberry. J. Carbohydr. J. Biotechnol. A.. Bioeng. L. 5. T. Jung. Li. M. Biotechnol.R. 1. 8. B. Felby. Adney and J. Biochem. N. Mitchell and G. B.. Appl. Appl. Ruiz. Biores. Assessment of Bermuda grass and bunch grasses as feedstock for conversion to ethanol.N. Casler. Templeton.. 290–300.E. Agric. L. Schols. Fukushima and R.K.G. 5–16 (2003). G. A. 2005. P. D. B. Dien. H. Wyman.S. Dale and M. 137–149 (1996). and neutral degradation products in biomass pretreatment hydrolysates. Determination of protein content in biomass. Standard assays do not predict the efficiency of commercial cellulase preparations towards plant materials.J. Voragen and H. Bioprod. Determination of insoluble solids of pretreated biomass material.G. Jørgensen.D.

M. Laboratory Analytical Procedure. Holtzapple and M. Zacchi. World J. Biochem. Progress and challenges in enzyme development for biomass utilization. Total dietary fiber determined as neutral sugar residues. Lopez. Optimization of pH controlled liquid hot water pretreatment of corn stover.D. R. 301–304 (1998). 53–67 (2003). Newman and Q. Lloyd and C. Biores. Biores. Pettersson. 846–850 (2007). Laboratory Analytical Procedure. Himmel.. Appl. Laboratory Analytical Procedure. J. Cherry. Ladisch. Microbiol.. Biochem. Determination of sugars. Nguyen. B. 673–686 (2005a). K. Comparative sugar recovery data from laboratory scale application of leading pretreatment technologies to corn stover. H. M. 23. D. 1986–1993 (2005b). R. P. 1967–1977 (2005).A. E.. Q. C.J. Balan. Sluiter. Theander. Schell. Sedlak and M. R. Chundawat. Biotechnol.. Bals. 45. Lloyd and C. Strokes and D. B. Biotechnol. Thammasouk. W.H. M.. Tucker. Appl. Ruiz.E.P. DC.N. Technol. R.E. Nieves. Templeton. Stenberg. Nichols.. C. N. Determination of structural carbohydrates and lignin in biomass. Biores. M.. Isolation of microorganisms for ´ biological detoxification of lignocellulosic hydrolysates. Appl. Aman.A. 437–443 (1997). Eng. R. O. R. Effects of temperature and moisture on dilute-acid steam explosion pretreatment of corn stover and cellulase enzyme digestibility. Galbe. Dilute acid pretreatment of softwoods. A. F. Wright. Hahn-Hagerdal.. Elander. Food Chem. . Graham. USDA Economic Research Service. DC. B.C. R. 96. M. 96. NREL/TP-510-42623 (2006).L. 96. Hames. survey. Wyman. Optimization of ammonia fiber expansion (AFEX) pretreatment and enzymatic hydrolysis of Miscanthus x giganteus to fermentable sugars. Westerlund. 165–178 (2003). 14. Hendrickson. Mosier.J. C. 96. Tengborg and G. and degradation products in liquid fraction process samples.E. NREL/TP-510-42619 (2005a). Biochem. S. (2005). P. Turhollow. Biotechnol. Ladisch. Keller and D. K. Tandjo and M. Adv. M. 58. Technol. Biotechnol. B. Scarlata. N. Dale. Templeton and D.F. Sluiter and D. Sluiter. 125–131 (2004). Elander and M. C. J. USDA and DOE. Perlack. R. Tucker. 105–108. Scarlata. Z. C. D. Mosier. Farmer.. C. and analysis of commercial cellulase preparations suitable for biomass conversion to ethanol. NREL/TP510-42618 (2005b).E. byproducts. Sluiter. M. M.D. Y.H. Dale. Lee. Larsson. Kim. Features of promising technologies for pretreatment of lignocellulosic biomass.A. B.T. B. Dilute-sulfuric acid pretreatment of corn stover in pilot-scale reactor. N. V. Wyman.A. Bothast. Sluiter.C. A.. 108. Nguyen. Combined sugar yields for dilute sulfuric acid pretreatment of corn stover followed by enzymatic hydrolysis of the remaining solids.T.. S. B.P. T. Washington. 1030–1044 (1995). Crocker.J. Murnen. Templeton.K. L. Biochem. A. Westcott.E.I.J.E. Technical communication. 64. L.. Moreno and R. Elander. 70–72. Ruiz. Biotechnol. Appl. Lee.J. Palmqvist. 78. Dale. Appl. J. E. J. uronic acid residues and Klason lignin (The Uppsala Method): collaborative study. Microbiol. Biotechnol.S.Mass Balances and Analytical Methods for Biomass Pretreatment Experiments 231 T. McMillan. Biotechnol. Wyman. J.S. USDA Rep FDS-07D-01 (2007). Boynton. Adney. Dien. Washington. Merino and J. 171–179 (1996).L.E. Agric.L. Szengyel. Biores.R. Holtzapple. 95–120 (2007). Wyman. R. Prog.T. Scarlata. N. Biochem. M. Application of a depolymerization model for predicting thermochemical hydrolysis of hemicellulose. Andersson and D. Ethanol expansion in the United States: How will the agricultural sector adjust?. Technol.Y. 69–85 (2003). Biotechnol.Biomass as feedstock for a bioenergy and bioproducts industry: the technical feasibility of a billion-ton annual supply. M. Sluiter and D.S. Ruiz. Biores.. Rep. J. Ehrman.Y. Technol. 2026–2032 (2005). D. da Costa Sousa and B. Technol. 77–87 (1998). K. Hames. 105. Schell. Newman and J. J. B. Determination of extractives in biomass. Ho. DOE/GO-102995-2135. AOAC Int. M. Erbach. C. B..P. Ladisch and Y. R. A. 105–108. M. Influence of extractives on the analysis of herbaceous biomass. R..A. Design and operation of a bench-scale process development unit for the production of ethanol from lignocellulosics. Penner.


[For a comprehensive review of pretreatment technologies. (2005). The major useful components of lignocelluloses for biofuels conversion are the polysaccharides cellulose and hemicellulose. ammonia fiber explosion. and annual plants such as crops of herbaceous angiosperms. mannose. . acid-catalyzed steam explosion. Hans Blaschek e and Hideaki Yukawa The contribution of Liu has been written in the course of his official duties as US government employee and is classified as a US Government Work. the reader should consult Chapters 10 and 11 of this book. Wyman et al. Lignocellulosic substrates are plant materials including softwood (gymnosperms). Cellulose is a high-molecular-weight polymer of hexoses. and J€rgensen et al. including dilute acid hydrolysis. Lewis Liu and Hans P. steam explosion. Blaschek 12.1 Introduction Overcoming the impact of inhibitory compounds derived from lignocellulosic biomass is one of the major challenges for a sustainable biomass-to-biofuels industry. and enzymatic hydrolysis. Unlike the easily available sugars that are utilized in starch-based fermentations. mainly glucose. Sun and Cheng (2002). Moisier et al. wet oxidation. (2007).12 Biomass Conversion Inhibitors and In Situ Detoxification Z. (2005). Nasib Qureshi. several pretreatment methods have been commonly applied. and some hexoses such as glucose.] Research and development in this area is under way in order to o Biomass to Biofuels: Strategies for Global Industries Edited by Alain Verts. and rhamnose. alkaline hydrolysis. hardwood (woody angiosperms). that can be further degraded into simple sugars and utilized by fermentative microorganisms. Hemicellulose is a polymer of mainly pentoses including xylose and arabinose. which is in the public domain in the United States of America. wet explosion. In this respect. lignocellulose biomass must be treated specifically to depolymerize and release simple sugars for microbial utilization. in addition to other reviews on the subject by Duff and Murray (1996). galactose.

HMF) can be formed by dehydration of these sugars. Antal et al. Many other aldehyde compounds and phenolic Cellulose Hexoses Dehydration HMF Levulinic acid Lignocellulosic biomass Hemicellulose Pentoses Furfural Formic acid Furoic acid Acetic acid Ferulic acid Lignin Other aldehydes Other phenols Other acids Figure 12. Pentoses and some hexoses are released from hemicelluloses. Saha. Antal et al. Acetic acid and other organic acids are also released from hemicellulosic fractions.. dilute acid hydrolysis is commonly used in biomass degradation for hydrolysis of the hemicellulose fraction and increased fiber porosity to allow enzymatic saccharification and fermentation of the cellulose fraction (Bothast and Saha. and practical feasibility. However.1 Schematic showing the degradation of lignocellulosic biomass into fractions of cellulose. cost-efficiency. 1997. Olsson and Hahn-H€gerbal. Ezeji et al. HMF and furfural can further break down to produce levulinic acid. 12. Hemicellulosic fractions of biomass hydrolysate contain various organic acids (Figure 12. mainly by dehydration of sugars and degradation of lignin fractions. 1991. 1985.. Furfural and HMF are considered to be the representative inhibitors of yeast and bacterial growth and fermentation (Chung and Lee. formic acid. 1996. there is as yet no universally ideal pretreatment procedure that takes into consideration energy requirements. Larsson et al. Lewkowski. 2007). from which inhibitory compounds are generated during the biomass pretreatment process .. 2000. 1948.1). However. Further degradation of hexoses released from cellulose can lead to more HMF formation under high temperature and acidic conditions (Dunlop.. 1990. from which 2-furaldehyde (furfural) and 5-hydroxymethyl-2-furaldehyde (5-hydroxymethylfurfural. 2001). a major limitation of this method is the generation of numerous byproducts and compounds that inhibit microbial growth and fermentation. Each of the currently available methods has different advantages and disadvantages. One commonly observed problem is the generation of inhibitory compounds of various byproducts during the pretreatment.234 Ethanol and Butanol develop methods that limit the production of potential inhibitors produced during the deconstruction of biomass. Taherzadeh a et al. 1999b. 2003). and furoic acid.. hemicellulose and lignin.2 Inhibitory Compounds Derived from Biomass Pretreatment For economic reasons.

it is understood that specific chemical functional groups are responsible for the inhibitory effect and toxicity to microbes. However.. 1986. Aldehyde inhibitors are compounds with one or more functional aldehyde groups.. J€nsson o et al. furan inhibitors. 1984. Larsson et al. The variety and concentration of inhibitory compounds also depend upon the pretreatment conditions such as treatment materials. pressure. Klinke et al. Moreover. identification. The inhibitor and toxic effects appear to be caused by the aldehyde functional group rather than the furan ring. Naming the inhibitors by functional group implies likely mechanisms of the inhibition. Liu et al.. each containing a furan ring and an aldehyde functional group (Delgenes et al. Buchert et al. these additional steps add cost and complexity to the process and generate extra waste products (Martinez et al... Sierra-Alvarez and Lettinga. 2003. Tran and o Chambers. 1986. 2002. including industrial strains. regardless of the base structure of a furan ring. phenols.. this class of compounds includes inhibitors such as furfural and HMF. Mussatto and Roberto. and other compounds having .2). temperature.. 2000. are also furan derivatives. 1999). In general. ketones... and time duration. pH. Ranatunga et al. 1985. furfural and HMF are furan derivatives and commonly called ‘furan inhibitors. Although more than 100 compounds were detected as potential inhibitors from biomass hydrolysates (Luo et al. 1999.. chemical. Taherzadeh et al. the degradation of byproducts produced during the fermentation can vary significantly. For example. a benzene ring or a phenol-related structure. In order to facilitate fermentation processes. Fenske et al. vanillin (Ando et al.. 1980.. but less toxic to fermentative microorganisms (Liu et al. Tran and Chambers. 1998. 1990. Clark and Mackie... Numerous inhibitors have generally been recognized as weak acids. This property could perhaps be ascribed to an easier transport of the smaller molecules via a variety of mechanisms. Klinke et al.. 2004. Baquinero et al. 1990. 1990. Ranatunga et al. common name.. 2002). 2004). Martin et al. 1999). In addition. J€nsson et al. syringaldehyde (Buchert et al. 1996. and molecular weight (MW) (Figure 12. are susceptible to the complexes associated with dilute acid hydrolysis pretreatment (Palmqvist et al. 1985. 1991). in this chapter we present a classification of inhibitors based on their chemical functional groups as aldehydes.. 1999.. Liu et al. 2008b) (see below for more detailed discussion on this subject). Each inhibitor is presented with a chemical structure. or biochemical detoxification procedures – are often required to remove these inhibitory compounds.. 1997b). 2002). 2000. 2004). 1994). metals and SO2 inhibitors resulting from hydrolytic equipment and additives can also be harmful to microbial growth and metabolic activities. and organic acids. the synergistic effects of inhibitory compounds in a mixture are clearly beyond a simple sum of the individual compound effects. Larsson et al. With increased knowledge and understanding of the mechanisms of inhibition and detoxification.. 1997a.’ Evidence has shown that the metabolic conversion products of furfural and HMF. Therefore. For example.. 1998. Other aldehyde inhibitors include 4-hydroxybenzaldehyde (Ando et al. Due to the heterogeneous nature of lignocellulosic biomass. Buchert et al.. and potentially helps to facilitate the investigation and understanding of the detoxification of the inhibitors.. many have not been well studied.. 1984. furan methanol (FM) and furan-2.5-dimethanol (FDM). additional remediation treatments – including physical. it was observed that low-molecular-weight compounds show more toxic effects to microbes than do high-MW compounds (Clark and Mackie. Most yeasts.Biomass Conversion Inhibitors and In Situ Detoxification 235 compounds are generated from lignin degradation (McMillan. including passive diffusion. and phenolics.. 1999b. Fenske et al.

11 O 4-hydroxy-3-methoxybenzaldehyde (vanillin) MW 152. ketones. HMF] MW 126.15 1-(4-hydroxy-3-methoxyphenyl)ethanone (acetovanillin) MW 166.15 (coniferyl aldehyde) MW 178.2 A classification of compounds inhibitory to fermentative microorganisms derived from biomass pretreatment based on chemical functional groups including aldehydes.17 O Ketones O O OH O OH O OH 1-(4-hydroxyphenyl)ethanone (4-hydroxyacetophenone) MW 136.16 O OH O OH O O OH O OH O 3-hydroxy-4-methoxybenzaldehyde (isovanillin) MW 152.15 O 2-hydroxy-3-methoxybenzaldehyde (2Z )-3-(4-hydroxy-3-methoxyphenyl) (ortho vanillin) -prop-2-enal MW 152. The molecular weights are shown. with common names in parentheses .18 4-hydroxy-3.15 (2E )-3-phenylprop-2-enal (cinnamaldehyde) MW 132.5-dimethoxybenzaldehyde (syringaldehyde) MW 182.5-dimethoxyphenyl)ethanone (acetosyringone) MW 196.09 O O 5-(hydroxymethyl)furan-2-carbaldehyde [5-(hydroxymethyl)furfural.2 Figure 12. organic acids and phenols.17 1-(4-hydroxy-3.236 O O O Aldehydes O O O O OH 4-hydroxybenzaldehyde (HBA) MW 122.12 OH HO O Ethanol and Butanol Furan-2-carbaldehyde (furfural) MW 96.

4-diol (hydroquinone) MW 110.14 3-methylbenzene-1.2-diol (methylcatechol) MW 124.2 (Continued) 237 .11 benzene-1.4-diol (2.2 OH OH O OH OH O O O OH 4-(hydroxymethyl)-2-methoxyphenol (vanillyl alcohol) MW 154.2 Figure 12.14 2-methoxyphenol (guaiacol) MW 124.2-diol (ethylcatechol) MW 138.2 OH 2.2-diol (catechol) MW 110.Phenols OH HO OH HO OH OH OH benzene-1.6-dimethoxybenzene-1.14 2-methoxy-4-(prop-2-en-1-yl) phenol (eugenol) MW 164.16 O OH 4-[(1E )-3-hydroxyprop-1-en-1-yl]-2 -methoxyphenol (coniferyl alcohol) MW 180.11 OH O OH OH OH O OH 2-methylphenol MW 108.11 4-ethylbenzene-1.16 Biomass Conversion Inhibitors and In Situ Detoxification 2-methoxy-4-[(1E )-prop-1-en-1-yl] Phenol (isoeugenol) MW 164.6-dimethoxy-hydroquinone) MW 170.16 phenol MW 94.

12 O OH HO 4-hydroxy-3.15 3.17 Hydroxy(4-hydroxy-3-methoxyphenyl) (2E )-3-(4-hydroxy-3.17 MW 164.12 3.17 MW 224.5-trihydroxybenzoic acid (gallic acid) MW 170.4-dihydroxybenzoic acid (protocatechic acid) MW 154.2 (Continued) .16 O OH HO O OH O OH O OH O OH O OH O OH (2E )-3-(4-hydroxy-3-methoxyphenyl) -prop-2-enoic acid (ferulic acid) MW 194.5-dihydroxybenzoic acid Mw 154.05 O O O OH OH OH HO OH OH OH OH O OH OH Formic acid MW 46.4.5-dimethoxyphenyl) -acetic acid -prop-2-enoic acid (guaiaclyglycolic acid) (sinapic acid) MW 198.12 2-hydroxybenzoic acid MW 138.18 (4-hydroxy-3-methoxyphenyl) -acetic acid (homovanillic acid) MW 182.Organic Acids O O O O O OH OH O OH OH O 238 OH Acetic acid MW 60.12 2.12 Hexanoic acid (caproic acid) MW 116.03 Furan-2-carboxylic acid (2-furoic acid) MW 112.16 O OH Ethanol and Butanol OH 4-hydroxybenzoic acid MW 138.12 3-hydroxybenzoic acid MW 138.5-dimethoxybenzoic acid (2E )-3-(4-hydroxyphenyl)-prop-2-enoic (syringic acid) acid (4-hydroxycinnamic acid) MW 198.08 4-Oxopentanoic acid (levulinic acid) MW 116.12 O OH O O OH OH O OH O OH OH HO O O OH OH OH 4-hydroxy-3-methobenzoic acid (vanillic acid) MW 168.21 Figure 12.

many previously recognized phenolic compounds are now grouped as members of the organic acid inhibitor class based on their functional structure. 1996. J€nsson et al. 1986. 2007).. Larsson et al. Zaldivar and Ingram.. gallic acid. Klinke et al. 2000). furoic acid (Klinke et al.. Barquinero et al. Baquinero et al. 2-hydroxybenzoic acid o (Ando et al. Tran and Chambers. 1999a). Zaldivar and Ingram. 2000). 1990. Fenske et al. Klinke et al. formic acid (Ranatunga et al. 2-methylphenol.. J€nsson et al. 4-[(1E)-3-hydroxyprop-1-en-1-yl]-2-methoxyphenol (coniferyl alcohol). benzene-1. 1997. Klinke et al. 1999a). Buchert et al. protocatechic acid o (Larsson et al. Individual strains have been isolated that retain their ability to produce ethanol in the presence of 10 to 79 mM of either furfural or HMF.. and sinapic acid (Baquinero et al.. syringic acid (Ando et al.. These inhibitors are thought to be exert their inhibitory actions via their carboxyl functional groups. 2002)... 1986. J€nsson et al.. 1999.. 1998).... Buchert et al. 2001. 1998. 1980. 1984.. 3-methylbenzene-1.2-diol (methylcatechol)... 2002.3 Inhibitory Effects The effects of different inhibitors vary widely among different strains of yeast and bacteria (Beall et al.. 1985). 1980. 2003. Talebnia et al. 1997a..5-dihydroxybenzoic acid (J€nsson et al. 1999). 1991. Tran and Chambers. Martin et al.. Klinke et al... ortho-vanillin (Larsson et al... 2003. 2002. . 2-methoxyphenol (guaiacol)..2-diol (ethylcatechol). 1998.. 2-methoxy-4-[(1E)-prop-1-en-1-yl] phenol (isoeugenol). 4o hydroxycinnamic acid (Ando et al. vanillic acid (Ando et al.. 1998).. and coniferylaldehyde (Buchert et al. 4-(hydroxymethyl)2-methoxyphenol (vanillyl alcohol). 1999).. 1997b. furfural and HMF inhibit cell growth and ethanol production rates at lower concentrations. Moreover.. o 12.. 1999). 1986). 4-ethylbenzene-1.. 1986. 1986.. inhibitors sharing a common carboxylic acid functional group are now collectively classified as organic acid inhibitors. 2002). 1998. Larsson o et al.. 1999). 1984.. 3-hydroxybenzoic acid (J€nsson et al. 2002). 1999a). Notably..2-diol (catechol) (J€nsson et al.Biomass Conversion Inhibitors and In Situ Detoxification 239 a benzene ring or a phenol-based structure including isovanillin (Larsson et al. 1990.. Tran and Chambers. Fenske et al. 2000).. 1986. 2. 2005. 4-hydroxybenzoic acid (Ando et al. 1999). The remaining phenol-based inhibitors are grouped together including phenol (Clark and Mackie. 1980). 1999). This class of compounds includes simple acids as well as furoic acid with a furan ring that was previously considered as being a furan inhibitor. Klinke et al.. Ketone inhibitors include 4-hydroxyacetopheone and the closely related compounds acetovanillone and acetocsyringone (Klinke et al. 2002. Cinnamaldehyde is another aldehyde inhibitor typically present in lignocellulosic biomass hydrolysates. Fenske et al. benzene-1. and 2.. levulinic acid (Zaldivar et al... Larsson et al. Delgenes et al. Klinke et al. Baquinero et al. Similarly. Klinke et al. Klinke et al. 1999. 2003)... 1990). Larsson et al. ferulic acid (Klinke et al. Clark and Mackie. 2-methoxy-4-(prop-2-en-1-yl) phenol (eugenol)... caproic acid (Ranatunga et al... Sakai et al. guaiaclyglycolic acid (Buchert et al. o 1998. 2002).6-dimethoxy-hydroquinone) (Clark and Mackie. Inhibitory compounds of this class all contain a carboxyl functional group and include acetic acid. Klinke et al. Ranatunga et al. homovanillic acid (Larsson et al.. Ezeji et al. 1985... 1999. 2007.4-diol (hydroquinone) (Larsson et al.6-dimethoxybenzene-1. 2003. 1980.. 1999). 1990.4-diol (2. 1984.. 2001.. Zaldivar et al. these compounds all share a common ketone functional group.

Palmqvist et al.. and metabolic conversion activities (B and D) of S. and glucose to ethanol are significantly delayed in the presence of inhibitors as compared to a control culture. 1999). 2004). Dose-dependent inhibition effects of furfural and HMF were particularly characterized for the yeast S. 1997b. Candida shehatae. 2000. Ranatunga et al. and no biological activity or HMF transformation is observed.9 0. Pichia stipitis..240 Ethanol and Butanol including strains of the following species: Saccharomyces cerevisiae. metabolic conversion activities in transformation of HMF to FDM. However. Sakai et al. Liu et al...Y.3 0. cerevisiae NRRL Y-12632 in response to 5-(hydroxymethyl)-2-furaldehyde (HMF) challenges at 30 mM (A and B) and 120 mM (C and D).0 0 1. furfural to FM. in the presence of a high concentration of a single inhibitor such as 120 mM HMF.2 30 (A) g/L mM 20 (B) OD600 0.. Furthermore.0 0 20 40 60 80 100 120 140 75 50 25 0 0 20 40 60 80 100 120 140 Time (hour) Time (hour) Figure 12.5 1. Lee et al. Most notably. and Escherichia coli (Delgenes et al. Y.3C and D). yeast demonstrate dose-dependent cell growth and metabolic conversion activities in response to varied doses of HMF and/or furfural (Liu et al.. cerevisiae (Taherzadeh et al. Corynebacterium glutamicum.. Ranatunga et al.. Zaldivar and Ingram. are susceptible to these inhibitors. Most yeasts.3 Cell growth (A and C) as measured by absorbance at OD600.. 2007..5 1. 1996. When added at a concentration of 30 mM to a yeast culture. Zymomonas mobilis.9 0. and furan dimethanol (4). Figure legends of HPLC assay data (B and D) labeled as glucose (*). biotransformation. 2005. ethanol (*).6 0. yeasts are completely inhibited and no cell growth is observed even after 128 h incubation (Figure 12. the major effects of these inhibitors comprise an extended lag phase of both growth and metabolic activities (Figure 12. including industrial strains. On a defined medium under controlled conditions. 2004). 1999.3A and B). Talebnia et al. HMF (~). synergistic repression is commonly observed when a combination of inhibitors or inhibitor complexes 1. the remaining values are units of mM . 1999. 1997a. The cells appear to be completely repressed at this inhibitor concentration.2 20 40 60 80 100 120 140 10 0 0 20 40 60 80 100 120 140 (C) g/L mM 125 100 (D) OD600 0.6 0.3 0. Glucose and ethanol are given in g lÀ1.

1984.. this can perhaps be best exemplified by the observation that. and (3) syringyl (Ando et al. 1996.. ranked from strong to weak. Organic acids. While demonstrating mild inhibition to most yeasts and bacteria. Klinke et al. 2004). 1986. Phenolic compounds cause increased membrane fluidity and affect membrane permeability (Heipieper et al. 1997b. Lee et al. However... Klinik et al. The order of inhibitory effects. 2007.L. Other common organic acids such as 4-hydroxybenzoic acid and vanillic acid cause inhibition of growth and ethanol production at relatively low concentrations (Ando et al.. Clark and Mackie. The degree of inhibition on cell growth and ethanol production also varies depending on the strains tested. 1986. Ranatunga et al.. 1945). 2000). some acids such as 4-hydroxycinnamic acid and ferulic acid can severely restrict ethanol productivity by yeast at low concentrations (Larsson et al. guaiacyl. Other aldehyde inhibitors. 2003). 1999). Inorganic salts produced from the biomass process and heavy metal ions such .. 1999). 2007. Cell walls and membranes of yeast cells grown under furfural and HMF-challenged conditions appear damaged when compared to those of controls grown in the absence of any inhibitor (S. 1996. 1999).. (2) guaiacyl. 1986. Delgenes et al.. 2003. and syringyl.. yeast cells can be killed even at low concentrations (Liu et al.. 1999. 1994. Lee et al.. Zaldivar et al.. Ezeji et al. 2001. and coniferyl alcohol almost completely inhibit E. The three main phenol structure building blocks in lignin are described as p-hydroxyphenyl. Khan and Hadi. hydroquinone. Zaldivar et al. syringic acid is extremely toxic to Clostridium beijerinckii even at very low concentrations (Klinke et al. Ranatunga et al.. including phenol aldehydes such as 4-hydroxybenzaldehyde. 2001.. aldehydes and phenols are more toxic than organic acids (Leonard and Hajny. 1999. 1999). 2002). Klinke et al. 1984.. Moreover. 2004).. 2001. 2007. unpublished results).. 1997a. 1997b. 2000. Ezeji et al.. Larsson et al. coniferyl aldehyde. but are relatively less toxic to yeast (Larsson et al. However. 2003). in terms of reduced growth and ethanol yield (Klinke et al. Gorsich and Z.. 1999. 2000). cell growth is delayed and ethanol productivity significantly reduced. 2000). Palmqvist et al. Delgenes et al. Zaldivar et al... 1994). Liu.. 1999). syringaldehyde and vanillins. Ranatunga et al. in the presence of a combination of inhibitors. As a result. were observed to be inhibitory at less than 10 mM to most yeast and bacterial strains (Ando et al. Palmqvist et al. Modig et al.. 2003. 1999.. suggesting the involvement of a hydrophobic target such as the cell membrane (Zaldivar and Ingram. 2003. In general. Such alterations when combined may enhance synergistic inhibition.. is as follows: (1) hydroxyphenol.. and to inhibit protein and RNA synthesis (Sanchez and Bautista.. in general....W. Klinke et al. These inhibitors are reported to reduce enzymatic biological activities. 1988. 1999. coli (Zaldivar et al. Phenols such as cathecol. 1999... are more toxic to isolates of bacteria than yeasts.. to break down DNA. Sakai et al. aldehyde inhibitors derived from lignin degradation appear to be more inhibitory than those derived from sugar dehydration (Lee et al.. 1997b. The general toxicity of a phenol compound to yeasts has been correlated to the degree of its methoxy substituents ortho to the phenol hydroxyl group. Ketones appear to exert a greater inhibitory effect on bacteria such as Thermoanaerobacter mathranii than on yeasts. eugenol and isoeugenol are inhibitory to yeasts at low concentrations. Klinke et al... Lee et al.Biomass Conversion Inhibitors and In Situ Detoxification 241 is added. Zaldivar et al. Clark and Mackie. The toxicity of organic acids has been correlated with their degree of hydrophobicity. These chemicals differ in their methoxygroups ortho to the phenol group (Klinke et al. Ranatunga et al.

including furfural and HMF. In reality.and cation-exchange resins has been reported to result in better detoxification results and improved fermentability when compared with other methods (Gong et al. and copper that originate from the corrosion of hydrolysis equipment can also be inhibitory to microorganisms (Mussatto and Roberto. 2003). vanillin. The application of both anion. Nilvebrant et al. and improves microbial growth and fermentation performance (Martinez et al. Lee et al.. Inhibitory compounds also vary according to each specific strain of fermentative microorganism utilized... Larsson et al.. unsatisfactory performance resulting in increased inhibition was also observed using such treatment. This additional removal step complicates the processing procedures. inhibitory compounds present in a hydrolysate have synergistic inhibitive effects over that of the sum of individual toxic effects. chromium. Vacuum evaporation is a physical method that is used to reduce the amounts of volatile compounds present in different hydrolysates.Y. Palmqvist and Hahn-Hagerdal. and also generates additional waste. This method in general reduces aldehyde and ketone inhibitors. Klinke et al.. 12.. 1993. diatomaceous earth has been used to absorb undesirable compounds (Ribeiro et al. 1991).. 1999. For example. Larsson et al. Palmqvist et al. Several chemical methods have been applied to precipitate toxic compounds such as alkali treatment using Ca(OH)2 or NaOH. 1999. the concentrations of furfural.. Larsson et al. 1998. 1997. the removal of inhibitory compounds from hydrolysates is typically necessary to facilitate efficient microbial growth and fermentation (Mussatto and Roberto. 1986. 2004). Silva et al. There again.. 2001). the efficiency of inhibitor removal varies according to the source of the hydrolysate. However. and acetic acid in test hydrolysates were reported to be significantly reduced from 29 to 100 % following such evaporation treatment (Converti et al. the type of inhibitory compounds present in a hydrolysate depends upon the types of pretreatment and biomass materials utilized in the process. An obvious disadvantage of this method is that it generates a CaSO4 precipitation product that must be removed.242 Ethanol and Butanol as iron. increases cost. Rodrigues et al. This was mainly ascribed to that this method concentrates the non-volatile toxic compounds as well.G. On the other hand.. 2000. 1999a. removal of only the major inhibitors often results in significantly improved microbial growth and fermentation (Bucher et al.4 Removal of Inhibitors Since biomass conversion inhibitors can be problematic for various fermentative microorganisms. or biological in nature. 2000a. 2001. the level of the toxic compounds can be reduced by applying activated charcoal for attaining improved microbial fermentation performance (Domiguez et al. nickel. Roberto et al. Notably. 1999a. Similarly. W. Silva and Roberto. The most commonly employed inhibitor removal methods are physical. 1999). Lee et al. this approach . the pH of the hydrolysate can be increased to 9–10. and subsequently readjusted to an appropriate value using acid addition prior to microbial fermentation. 1996. However. Tran and Chambers... However. 2001). 1999). By employing this overliming treatment. fermentative microorganisms have been observed to be inhibited by concentrated non-volatiles such as lignin derivatives and extractives (Parajo et al.. chemical. 1990. 2000b.. 2004). 2001). Nonetheless.. 1996... Y. Palmqvist and Hahn-Hagerdal. increases energy requirements..

improved hydrolysate fermentation by adaptation of fermentative microorganisms to hydrolysates has been reported (Olsson and Hahn-Hagerbal. continuing efforts to identify and understand the profiles of inhibitory compounds present in various hydrolysates remains a critical area of research for enabling the development of improved detoxification methods. the removal of inhibitors from hydrolysates using the above mentioned methods may not be an economically worthwhile approach given the costs associated with additional processing steps and the loss of fermentable sugars. 1996. many of the industrially interesting microorganisms obtained thus far are not robust enough to withstand the stress conditions associated with the biomass conversion process. and evaporation (Alves et al. 2001). In addition. It is possible that some undiscovered compounds have synergistic inhibitory effects even at low concentrations. Parajo et al.. Converti et al. not all potentially inhibitory compounds have been identified to this date. inhibitor-tolerant strains of ethanologenic S. Liu et al. a further improved tolerant yeast strain designated NRRL Y-50049 was generated that withstands the synergistic inhibition caused by inhibitor complexes.. In contrast. 2000. Silva and Roberto. 1998). enzymatic treatment using peroxides and laccase obtained from the ligninolytic fungus Trametes versicolor has been reported to improve ethanol productivity of a fermentation process based on a willow hemicellulosic hydrolysate (J€nsson o et al.5 Inhibitor-Tolerant Strain Development The economics of fermentation-based bioprocesses for biofuels production rely extensively on the performance of microbial biocatalysts in industrial applications. such as applying pH adjustments. 2005). While dose-dependent inhibition of yeast by furfural and HMF has been observed and characterized (Taherzaadeh et al. The soft-rot fungus Trichoderma reesei has been reported to be able to degrade inhibitory compounds in a hydrolysate after steam pretreatment (Palmqvist et al. Often. cerevisiae with enhanced ability to detoxify the inhibitor furfural or HMF have been developed through directed evolutionary engineering (Liu. 1999. 2006. Sene et al.. 2008b). noteworthily.Biomass Conversion Inhibitors and In Situ Detoxification 243 may not be practical due to its high cost. Recently.. However.. a combination of different inhibitor removal methods is more efficient than any single method alone to remove a variety of inhibitory compounds. 2000). Considering the need of keeping low process costs of commodity products such as ethanol. Liu et al. This approach removes phenolic monomers and phenolic acids and appears to involve oxidative reaction of low-molecular-weight phenolic compounds. is unable to grow in the presence of the HMF and furfural complexes. and as demonstrated by HPLC . Therefore. 2004). as is the case for the aldehyde inhibitors furfural and HMF. 12.. The parent strain. inhibitor removal is a very selective process and it is difficult to identify a standard process which provides satisfactory results for all substrates.. this strain has been observed to complete an ethanol fermentation cycle in 48 h (Liu et al. activated charcoal adsorption.. 2001. 1997). The mix of inhibitory compounds present in hydrolysates varies based upon the source of the biomass. on the other hand. 1998.. Therefore. 1998.. Nonetheless. The development of yeast or bacterial strains that can withstand the presence of inhibitors is one of the keys for developing a sustainable lignocellulosic biomass-to-biofuels industry. boiling. Interestingly.

Multiple types of mutation have been successfully induced as a result of the application of selection pressure on yeasts (Z.. Glucose was completely consumed and a normal ethanol yield obtained at or prior to 48 h. Liu et al.. the development of ethanologenic yeasts with desirable characteristics using directed evolutionary engineering appears to be a promising arena and can constitute a very useful alternative for improving microbial strain performance (Liu and Slininger. the HMF became completely undetectable while the conversion product of HMF. As a result. but rather grows readily and completes the fermentation following typical kinetics. cerevisiae. the tolerant yeast strain Y-50049 does not require any acclimatization to the presence of inhibitors.. The directed evolutionary engineering procedures under laboratory settings that have been previously described (Liu et al. another inhibitor-tolerant yeast strain was obtained via an adaptation method. 2006. a specific enrichment of the genetic background of ethanologenic yeast may be needed that can be achieved by introducing exogenous genes. Adaptation methods have a long history of use in the yeast utilization industry. Liu and Slininger. the process can be iterated and such adapted strains could be efficiently used for further genetic manipulation. Such methods significantly reduce the time that is necessary to obtain desirable strain characteristics through nature evolutionary adaptation of yeast cells. efficient xylose-utilizing strains of S. Fed-batch and increased inoculum size are two important conventional methods that have been used to overcome the inhibitory effects of furfural and HMF. Recently. strain Y-50049 grows well on such media and dramatically reduces furfural and HMF. different populations with varied phenotypes can be recovered from a single recombinant strain under selection pressure (Sonderegger and Sauer. Tessier et al. . These results indicate that it is possible to in situ detoxify furfural and HMF when using the ethanologenic yeast S. Nichols et al. Nilsson et al. which was able to grow in a medium obtained by diluting to 50 % a sugarcane bagasse hydrolysate containing inhibitors (Martin et al. At 32 h. The basis of success of these simple methods depends upon the innate genetic potential of the yeast being engineered combined with appropriate selection procedures. unpublished results). 2005. On the other hand. 2004. which suggests that genomic adaptations occur during the laboratory evolutionary selection (Liu.. However. Persistent gene expression pattern shifts have been observed in the ethanologenic yeast under HMF challenge conditions.. most of these isolates are not capable of ethanologenic fermentation. Petersson et al. reached its peak concentration. along with ethanol. 2005). 2006). 2005). 2003). Empirical data suggest that. Sanchez and Bautista. since no strains have been available to grow in the presence of these inhibitors (Chung and Lee. This process primarily takes place during the lag phase of growth. cerevisiae were obtained through directed evolution after introduction of a single exogenous xylose isomerase gene (Kuyper et al.L. The furfural was completely depleted at 15 h as measured by HPLC assays.244 Ethanol and Butanol measurements... 1985. when using an evolutionary engineering method.. 2005). 1998. Selection under pressure is an evolutionary process of Nature. However. FDM.. Obviously. 1988. 2006). 2007). The typical inhibitor conversion products furan methanol (FM) and FDM were detected at the end of the fermentation. For example. The screening of microorganisms tolerant or able to utilize inhibitory compounds as a carbon source have been reported (Lopez et al. Additional studies in this area are expected to result in strains with significantly improved inhibitor tolerance. 2005) provide an easy and practical approach that can be extended to a broad range of applications.

an FDM preparation procedure has recently been described that can be used to prepare the necessary FDM standards for conducting by HPLC-based metabolic profiling analyses of HMF degradation (Liu et al. but rather a reduction of the aldehyde into alcohol. On the other hand. The mechanisms of the detoxification of furfural and HMF by yeast cells are unlikely to be involved in either the utilization or the degradation of the furan compound. Furfural and HMF are furan derivatives that comprise a furan ring and an aldehyde functional group with a composition of C5H4O2 and C6H6O3.. Based on the furfural conversion route.5-bis-hydroxymethylfuran (Figure 12. The chemical structure of the metabolite has been identified as that of a compound with C6H8O3 composition and a molecular weight of 128 Da. retain the furan rings and an alcohol group which replaced the aldehyde structure (Figure 12. 2004).4). FM and FDM – the chief conversion products of furfural and HMF – are accumulated at high levels in the fermentation medium by the tolerant strains. respectively with a composition of C5H6O2 and C6H8O3. knowledge of the HMF pathway has remained limited because there is no readily available commercial source for any of the HMF degradation products. 2008b).. 1999. 4-hydroxycinnamic acid was found to be converted by yeast cells into styrene (vinylbenzene) (Larsson et al.. 2001a). FM and FDM are less toxic to microbes. This limitation makes it virtually impossible to study HMF conversion mechanisms..Biomass Conversion Inhibitors and In Situ Detoxification 245 12..6 Inhibitor Conversion Pathways The furfural conversion pathway to FM by yeasts has been described (Morimoto and Murakami. 1967. Apparently. dihydroferulic acid [3-(4-hydroxy-3-methoxyphenyl) .. Taherzadeh et al. Liu... the use of ‘furan derivative’ as a general term for inhibitors such as furfural and HMF should be avoided. Therefore. 1992. Villa et al. A major metabolite of the organic acid ferulic acid has been identified as vinyl guaiacol (2-methyoxy-4-vinylphenol).5-dimethanol (FDM). The identification of FDM is an important development as it provides a basis for subsequent studies on the mechanisms of HMF inhibitor detoxification. 2006). Furfural can also be cleaved to form formic acid (Palmqvist and Hahn-H€gerdal. Moreover. at the end of the fermentation. a Unlike the well-studied furfural conversion pathway. HMF has a maximum absorbance at 282 nm. 2006) (Figure 12. Recently. 1991). the current hypothesis is that HMF is first converted into HMF alcohol (Nemirovskii et al. but the furan ring or associated alcohol functional groups are not (or are less) inhibitory. purified.4). Furthermore.. Following a vigorous investigative effort.4) (Liu et al. 2003. The NMR spectra thus obtained are consistent with that of a symmetrical molecule with a furan ring. the aldehyde functional group in furfural and HMF is toxic to yeast. An important clue to the symmetrical nature of the HMF degradation products was that the signals for the aldehyde proton and the asymmetric spectra of HMF were absent when the purified HMF-conversion product was analyzed using NMR. 1999. and FDM at 222 nm. FDM was further isolated from cell-free culture supernatants. As revealed by HPLC assays. also termed as 2. Sarvari et al. It is currently commonly accepted that furfural is first converted to FM and further reduced to furoic acid (Palmqvist et al. 2004. 2000b). an HMF metabolic conversion product was isolated and identified as being furan-2. Consequently. and characterized using mass and NMR spectra analysis (Liu et al. respectively. Liu. 1989). since the yeast does not appear to be inhibited for its growth and ethanol fermentation in the presence of these compounds. under oxygen-limited conditions. Nemirovskii and Kostenko. as FM and FDM are also furan derivatives. Their conversion products FM and FDM.

5-dimethanol (FDM) 4-oxopentanoic acid (Levulinic acid) Figure 12.246 O O OH Ethanol and Butanol Furan-2-carboxylic acid (2-Furoic acid) O O O + NAD(P)H OH + NAD(P)+ O OH 2-furaldehyde (Furfural) 2-furanmethanol (FM) Multiple Aldehyde Reductases O Formic Acid O HO OH O + NAD(P)H HO + NAD(P)+ O OH O 5-(hydroxymethyl)-2-furaldehyde (HMF) Furan-2.5-dimethanol (FDM) coupled with NADH and/or NADPH and catalyzed by multiple enzymes possessing aldehyde reduction activities .4 Conversion pathways of 2-furaldehyde (furfural) and 5-(hydroxymethyl)-2-furaldehyde (HMF) into 2-furanmethanol (FM) and furan2.

. 2005). a partially purified furfural reductase from E. cerevisiae is reduced during xylose fermentation when furfural is added to the medium (Wahlbom and Hahn-H€gerdal.. Likewise. 2006. In contrast. while alcohol dehydrogenase VI (ADH6) showed NADPH preference (Table 12. 2006).7 Molecular Mechanisms of In Situ Detoxification The biotransformation of furfural and HMF by yeast can be primarily ascribed to the action of NADH. 1999. 2008b).. furfural can cause an accumulation of acetaldehyde that results in a delay of acetate and ethanol production. 2006. Liu et al.. It is worth noting that recent studies on the reduction of furfural and HMF showed that mutants overexpressing a gene coding for a furfural and/or HMF reduction function have distinct cofactor preferences. Consistently. although the specific activity of this enzyme is higher with NADPH than with NADH (Table 12. the whole-cell protein extract from S. Similar observations have been reported for a crude protein extract that requires cofactor NADH for furfural reduction (Gutieerez et al. for example. In the presence of furfural. 2002). 2005. Nilsson et al.and NADPH-coupled enzymes (Palmqvist et al.1) (Liu et al. 2006). cerevisiae Y-50049 demonstrates that reduction activities of both furfural and HMF are coupled with either of these two cofactors. xylitol excretion by S. and therefore.. Consequently. Liu et al. For example. glycerol formation is reduced. 2002).. alcohol dehydrogenase VII (ADH7). a shortage of NADH is observed in yeast when cells are a incubated in the presence of furfural. which reflects the activity of a pool of the functional enzymes rather than that of a single gene product. With respect to the substrate furfural. and aldose reductase III (GRE3) exhibit on both substrates furfural and HMF a clear NADH preference. aldehyde dehydrogenase IV (ALD4). Studies performed at different ...1) (Petersson et al. cerevisiae was found that exhibited a NADH preference rather than NADPH (Nilsson et al. In contrast to the cofactor preference shown by these individually expressed genes..5). whereas in a later study a different strain of a S. HMF reduction by yeast cells was reported to have a preference for the cofactor NADPH (Wahlbom and Hahn-H€gerdal. the main trend of cofactor preference may be strain-dependent. 2008b). Similarly. coli by the same group demonstrated NADPHdependent activity on furfural (Gutieerez et al.. the ATP level is low and cell replication is thus limited. ADH6 appears to be less selective to either cofactor. This observation can be ascribed to the more diverse enzymatic activities displayed by the whole-cell extract of Y-50049. As a result. 2002. It appears that furfural reduction competes for NADH and interferes with cell glycolysis during the regeneration of NAD þ . either NADH or NADPH can be used for the aldehyde inhibitor reduction reactions. whereas dihydrocinnamic acid (3-phenylpropanoic acid) is produced from cinnamic acid (Figure 12. 2008b). 2002).Biomass Conversion Inhibitors and In Situ Detoxification 247 propanic acid] is produced from ferulic acid.. Petersson et al. Larroy et al. It has a been reported that reduced furfural tolerance was observed for selective deletion mutants of genes coding for significant enzymes involved with the pentose phosphate pathway (Gorsich et al. 2002). depending upon varied pathways and the composition of the functional aldehyde reductases involved. Most in vitro enzyme assays for HMF and furfural reduction were reported using wholecell protein extracts. 12. Furfural has been characterized as an electron acceptor (Wahlbom and HahnH€gerdal. Varied cofactor preferences were observed..

Under oxygen-limited conditions. (2001a) . and (2E)-3-(4-hydroxy-3methoxyphenyl) prop-2-enoic acid (ferulic acid) to 3-ethenyl-2-methoxyphenol (vinylguaiacol) catalyzed by phenylacrylic acid decarboxylase (Pad1p) and possible other enzymes. ferulic acid and cinnamic acid were converted into dihydroferulic acid (4-hydroxy-3-methoxyphenyl acetic acid hydrate) and dihydrocinnamic acid (phenylacetic acid).248 O OH Ethanol and Butanol O OH Phenylacetic acid (Dihydrocinnamic acid) (2E ) 3 h )-3-phenylprop-2-enoic l 2 i acid (Cinnamic acid) Ethenylbenzene (Styrene) Pad1p 3-ethenyl-2-methoxyphenol (Vinylguaiacol) OH O OH O O HO O OH (2E )-3-(4-hydroxy-3-methoxyphenyl) prop-2-enoic acid (Ferulic acid) OH (4-hydroxy-3-methoxyphenyl) acetic acid hydrate (Dihydroferulic acid) O Figure 12.5 Conversion pathways of (2E)-3-phenylprop-2-enoic acid (cinnamic acid) to ethenylbenzene (styrene). Adapted from Larsson et al. respectively.

(2008b) Liu et al.1 2. (2008b) Liu et al.5 3.5 157.3 13.Biomass Conversion Inhibitors and In Situ Detoxification 249 Table 12.7 78.7 0.1 3.6 5.7 — — — — — — 198 — — — — — — — — — — — — — — — — 100 25 26 105 184 158 175 156 133 37 60 46 7 135 — — — — 2.1 5.0 4. (2006) Present study Present study Present study Present study Present study Present study Present study Present study Present study Present study Present study Present study Present study Present study Present study Present study Furfural Furfural HMF HMF Furfural Furfural HMF Furfural HMF Furfural HMF Furfural HMF Furfural Furfural Furfural Furfural HMF Acetaldehyde Propanal Butanal Pentanal Hexanal Heptanal Octanal Trans-2-Nonanal Benzaldehyde Cinnamaldehyde Anisaldehyde Phenylacetaldehyde 190–210 — 6–8 — 62. (2008b) Liu et al.7 3. (2006) Petersson et al. (2008b) Liu et al. (2008b) Liu et al. (2008b) Liu et al.9 13.0 — — 86.2 353.6 — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — Relative activity measured as relative percentage to that of furfural reduction activity.8 7.4 0.4 0.7 8. (2006) Petersson et al.6 — — — 7. (2006) Liu et al.1 Specific activities and relative activities of whole-cell extract for selective overexpressed genes of Saccharomyces cerevisiae and Escherichia coli for reduction of furfural and 5-hydroxymethylfurfural (HMF) Enzyme Substrate Specific activity Relative Std (mU mgÀ1 Reference À1 (mU mg protein) activityà protein or %) NADH ADH6 ADH6 ADH6 ADH6 ADH6 ADH6 ADH6 ADH7 ADH7 ALD4 ALD4 GRE3 GRE3 FFR Y62 Y76 Y63 Y63 Y63 Y63 Y63 Y63 Y63 Y63 Y63 Y63 Y63 Y63 Y63 Y63 a à NADPH NADPH — 900–990 — $1300 — 97.1 9. (2006) Petersson et al.8 4. (2008b) Gutierrez et al.9 114. (2008b) Liu et al.3 21. .4 66.4 6. (2008b) Liu et al.1 157.3 Petersson et al.4 349.8 13.8 92.2 6.1 6.

This clearly represents an interesting area of development. the current practice of deleting the gene that code for GRE3 for strain improvement could thus potentially affect the comprehensive stress tolerance and detoxification ability of yeasts. 2002). 1998). A more comprehensive understanding of the role of GRE3 and its interactions among its corresponding functional enzyme group is needed in order to engineer strains with optimized detoxification and pentose utilization balance. 2008b) has only recently been observed. Nevertheless.250 Ethanol and Butanol laboratories have supported the observation that ADH6 exhibits an NADPH cofactor preference. As compared to the wild type. cerevisiae. As a result. 2005).. heat shock. 2008b). coli strain LYO1 (Table 12. 2008b). it impairs energy production. Therefore. 2003).to 100-fold more efficient than the corresponding oxidation reactions (Larroy et al. is probably unlikely to result in a dramatically increased susceptibility to either furfural or HMF. converting respectively HMF and furfural to FDM and FM while utilizing NADH as a cofactor (Liu et al.1) A partially purified protein of this enzyme demonstrated a strict NADPH . In particular. An enhanced expression of GRE3 is also observed under different stress conditions. alcohol dehydrogenase ADH7 shows significant reduction activities not only towards furfural and HMF when coupled with NADH. but also with other aldehydes such as cinnamaldehyde (Liu et al. respectively. and carbon starvation (Aguilera and Prieto. the enzymes ADH6 or ADH7 seem to act as aldehyde reductases and have similar substrate specificities toward various aldehydes (Larroy et al. Methylglyoxal is an aldehyde form of pyruvic acid.. cerevisiae. However. and kill cells of a wide variety of species (Kalapos. Despite aldehyde dehydrogenases having been known to play an important role in the acetaldehyde metabolism of yeasts (Aranda and del Olmo. It is interesting that kinetics studies indicated reductive reactions of ADH6 and ADH7 with various aldehydes and alcohol substrates are 50.. Kuyper et al. ALD4 is a major mitochondrial aldehyde dehydrogenase that is required for growth on ethanol and the conversion of acetaldehyde to acetate via equally utilizing NADP þ or NAD þ as coenzymes (Tessier et al. GRE3 is deleted to reduce undesirable xylitol production levels (Tr€ff a et al.. 1999). Likewise. overexpression of the ADH6 and ADH7 genes in ethanologenic yeast improves inhibitor tolerance as well as growth rates in the presence of furfural and HMF. GRE3 was recently reported to have strong a reduction activities to furfural and HMF (Liu et al. such as NaCl or H2O2 challenge. 2008b)... yeast clones overexpressing ADH6 and ADH7 show significantly higher reduction capabilities towards HMF and furfural (Liu et al. In fact. 2006). It is noteworthy that in numerous improved strains of S.. GRE3 also functions as an aldehyde reductase that also converts HMF and furfural to FDM and FM.. 2001). These improved characteristics can be directly attributed to the enhanced aldehyde reductase activities. Such an important property makes this enzyme an excellent candidate for efficient detoxification of inhibitors present in industrial biomass hydrolysates. overexpression of the GRE3 has been shown to increase methylglyoxal tolerance in S. and it has been reported to be an endogenous substrate of GRE3. it is a byproduct of metabolism that cannot be utilized by yeast cells. their potential for carrying out the detoxification of furfural and HMF during conversion processes of biomass to ethanol (Liu et al. GRE3 is an aldo-keto reductase that is involved primarily in the catabolism of xylose and arabinose (Tr€ff et al.. 2002). 2001. On the other hand... 2008b).. This enzyme was also found to function as a reductase. Furthermore. The activity of this enzyme is significantly increased when its gene is overexpressed in ethanologenic yeast (Petersson et al. A furfural reductase was reported to be instrumental in the reductive detoxification of furfural to furan dimethanol by the ethanologenic bacterium E. despite a single gene deletion. 2002). contributes to the generation of free radicals.

. investigations and advances in genomic biology have revolutionized our understanding and changed our view of yeast processing events. cerevisiae also improved yeast tolerance to phenolic inhibitors (Larsson et al. Among the 12 regulatory interactions identified using discrete dynamic system modeling studies. nor any susceptibility to either furfural or HMF under the controlled conditions (Liu et al. the overexpression of laccase from Trametes versicolor in S. crude whole-cell extracts of different clones overexpressing one of these genes exhibit significant aldehyde reduction activities to furfural and other toxic substrates (Table 12. it is unlikely that a single gene could play a decisive role in furfural or HMF detoxification. Liu et al. 2001b). the transcription factor Yap1p and Pdr3p are considered as being significant regulatory elements for HMF detoxification (Song and Liu. as previously mentioned. An illustrative diagram of ethanologenic yeast responses to inhibitor stress and corresponding detoxification pathways is presented in Figure 12. Phenylacrylic acid decarboxylase (Pad1p) catalyzes a decarboxylation step. 2008b). 2006). Therefore. Instead. For example.6. including functional genes and regulatory genes.. To date. and particularly aldehyde inhibitors detoxification pathways. the mRNA expression levels of a few recently identified genes of S. members of the pleiotropic drug resistance (PDR) gene family may play a significant role in coping with stress in order to promote cell survival (Liu et al.. but the detoxification of furfural and HMF by yeast apparently is performed by a complex metabolic network that is not limited to these genes. 2007). Interestingly. In addition to functional enzymes. none of the tested yeast mutants carrying a single gene deletion in either ADH6. a significant number of genes with enhanced expression in the presence of various inhibitors were also observed to share common transcriptional factors (Liu and Sinha. 2006). fewer than a dozen functional genes have been examined. 2006). 2008b). cerevisiae were observed to be significantly induced under both furfural and HMF challenges. several other enzyme activities are also likely involved in this conversion (Larsson et al. . and this approach will continue to be important in the future. has been proposed (Liu. In total.5). as well as regulatory cascades occurring among these genes. more than 300 genes have been identified as being differentially expressed under inhibitor stress conditions (Liu. However. As shown in a recent study. Genomic-based technologies will allow greater flexibility and power to design and develop more desirable and robust biocatalysts for achieving. 2001b). However. This clearly indicates that a single gene deletion in any of the above-mentioned genes does not significantly affect cell growth or tolerance to these inhibitors. however. the in situ detoxification of furfural and HMF likely involves multiple genes. 2006.Biomass Conversion Inhibitors and In Situ Detoxification 251 cofactor preference for furfural reduction activity (Gutierrez et al. 2000). Searching for similar activities in yeasts.1). It must be emphasized that this diagram remains largely incomplete. within the next decade. by which aromatic carboxylic acids are converted to the corresponding vinyl derivatives (Larsson et al. This was demonstrated during conversions of cinnamic acid and ferulic acid into styrene and vinylguaiacol (Figure 12.. ADH7. ALD4 or GRE3 demonstrated a detectable growth defect... A prototype of furfural and HMF conversion pathways relevant or critical to glycolysis and ethanol production. Notably. It is now clear that significant gene interactions and genomic regulatory networks need to be considered for achieving further improvement of the attributes of industrial strains. Single-gene studies have significantly contributed to our knowledge of gene functions during the past 50 years. 2006). a cost-effective and highly productive lignocellulosic conversion to ethanol..

The presence of aldehyde inhibitors appears to cause a redox imbalance that interferes with glycolysis. Taherzadeh et al. A shortage of the cofactor NADH has been observed in the presence of furfural (Wahlbom and Hahn-H€gerdal. Furfural is converted into 2-furanmethanol (FM) and HMF into furan-2. and potential interactions with other candidate genes in a yeast cell. and biosynthesis. .. A shaded upward arrow indicates upregulated or induced gene expression.6 Schematic diagram showing 2-furaldehyde (furfural) and 5-(hydroxymethyl)-2furaldehyde (HMF) conversion pathways relevant to glycolysis. On the other hand.252 Ethanol and Butanol Furfural HMF Vanillin Glucose Cinnamic acid Ferulic acid Glycolysis NAP+ ADP ATP Furfural NADH Pad1p Pyruvate Acetaldehyde NADH HMF PDR Gene Family NADPH NADP+ FM NAP+ Ethanol NAP+ FDM H+ CO2 Styrene NADPH NADP+ Vinylguaiacol 300+ Candidate Genes Multiple Aldehyde Reductases Vanillyl alcohol FM FDM Figure 12. aldehyde inhibitors could cause acetaldehyde accumulation that in turn would delay acetate and ethanol production. cell replication is limited. In the presence of these inhibitors. cinnamic acid and ferulic acid are respectively converted into styrene and vinylguaiacol..5-dimethanol (FDM) coupled with NADH and/or NADPH and catalyzed by multiple enzymes possessing aldehyde reduction activities. HMF. and other aldehyde inhibitors by yeast results in the formation of the corresponding alcohol coupled with the cofactors NADH or NADPH. The biotransformation catalyzed by multiple aldehyde reductases of furfural. catalyzed by phenylacrylic acid decarboxylase (Pad1p). 1999a. and glucose is not consumed until adequate furfural and/or HMF reduction levels are reached (Larsson et al. 2002). vanillin. while an open downward arrow indicates downregulated or repressed expression for members of the pleiotropic drug resistance (PDR) gene family and other candidate genes and thus further studies of microbial stress tolerance and in situ detoxification are needed to attain a sufficiently deep knowledge of these pathways to enable the construction of optimal cellular biocatalysts. HMF. Empirical evidence accumulated to this date tends to demonstrate that the regeneration of a sufficient NADH pool from NAD þ is an apparent requirement for achieving efficient cell glycolysis and the efficient reduction of furfural. As a result of this reducing equivalent a imbalance. 2000. Cinnamic acid and ferulic acid are converted into styrene and vinylguaiacol. the ATP level is typically low. and other aldehydes interferes. likely via the action of Pad1p and other enzymes. cell growth.

and organic acids. Notably. . ALD4. ADH7. 12. These reactions are catalyzed by phenylacrylic acid decarboxylase and most likely involve other enzymes. including ADH6. 2002. since NADPH is involved in numerous biosynthesis pathways. members of the PDR gene family are involved in the adaptive response to inhibitor stress conditions. comprising: (i) aldehydes. The organic acid ferulic acid is metabolized to vinyl guaiacol. HMF. Consequently. One major economic hurdle to creating a sustainable biomass-to-biofuels industry is that the removal of inhibitors by physical or chemical means is unlikely to be a cost-competitive practice.8 Perspective Based on their chemical functional groups. Many genes have been identified that code for enzymes possessing aldehyde reduction activities. A deeper understanding of inhibitor conversion pathways and mechanisms of in situ detoxification will undoubtedly facilitate the development of tolerant strains. many metabolic process may be significantly altered and delayed in the presence of these inhibitors. Members of the PDR gene family mainly code for membrane and transport-related proteins. The known mechanisms of the in situ detoxification of furfural. Despite a promising start. inhibitory compounds derived from lignocellulosic biomass pretreatment are classified into four groups. phenols. which is to design a detoxification process of lignocellulosic biomass that is as generic as possible in order to maximize flexibility in manufacturing operations. This implies one major difficulty. 2004). cinnamaldehyde. Furthermore. The development of tolerant strains by directed evolutionary adaptation under laboratory settings is expected to play a significant role when combined with the necessary enhancements of genetic background through recombinant engineering. Pdr3 and Yap1 are significantly involved in positively regulating gene responses and interactions during the coping reaction to HMF stress. thus impacting growth. Furfural is reduced to furan methanol and can be further catabolized to furoic acid and formic acid. Among the eight candidate transcriptional factors identified involving the inhibitor tolerance. Consequently. HMF is reduced into furandimethanol and further to formic acid and levulinic acid. to this date the ability to overcome inhibitor complexes in biomass hydrolysates remains a significant challenge.. dihydrocinnamic acid and dihydroferulic acid are also produced. the NADPH-coupled a furfural and HMF reduction activities of the whole-cell extract contribute to a great extent to in situ detoxification. in addition to several uncharacterized genes. As a result. Liu et al. (ii) ketones. one of the leading paths of development is to derive tolerant strains that are able to in situ detoxify harmful aldehydes. The mix of inhibitors – and thus the effects of inhibition on fermentative microbes – is of course strain-specific. while 4-hydroxycinnamic acid is decarboxylated to styrene. and GRE3. It appears critical to maintain a redox balance by reprogrammed pathways during the detoxification and ethanol production phases of the biomass-to-ethanol process. but varies largely depending upon the biomass sources used as a primary raw material. the latter are important as they are anticipated to play a significant role for yeast inhibitor tolerance and the regulation of detoxification gene interactions. and other aldehyde inhibitors are NAD(P)H-dependent aldehyde reductions catalyzed by multiple reductases.Biomass Conversion Inhibitors and In Situ Detoxification 253 Wahlbom and Hahn-H€gerdal. and (iv) organic acids. synergistic competition for NADPH during the reduction reaction adds additional stress on cells. (iii) phenols. Under oxygenlimited conditions.

39. Mechanism of formation of 5-(hydroxymethyl)-2furaldehyde from D-fructose and sucrose. Arai.P. Biotechnol. Aranda. Richards. 225–233 (1986).S. Genet. Converti.J.N. Chem. T. Bioeng. 199.L. Ferment. 82. Eng. Effects of lignocellulose degradation products on ethanol fermentations of glucose and xylose by Saccharomyces cerevisiae. I.. 91–109 (1990). Lee. Technol. 38. Perego. and G. Biotechnol. del Olmo. Identification of aromatic monomers in steam exploded poplar and their influences on ethanol fermentation by Saccharomyces cerevisiae. Xylitol production from hardwood hemicellulose hydrolyzate detoxification by Pachysolen tannophilus. Biotechnol. S. Res. . and S. Appl. I. 14. W. Bothast and B. Antal.. Perego. Enzyme Microb. J. Pretreatment of sugar cane bagasse hemicellulose hydrolyzate for xylitol production by yeast.. A. Leesomboon.J.. 44. Hanai. and H.A.. Prieto.S. Tsao. grant number 2006-35504-17359. Alves. 273–283 (2001). Appl. Parametric studies of ethanol production from xylose and other sugars by recombinant Escherichia coli.A.A. and J. Silva. J. K. Saha. Kiyoto. Richards. Appl. Silva. Tech.L. Appl Biochem. M.A. D. 261–286 (1997). Mok. Technol.S. 51. Dominguez.S. Process Biochem. Bioeng. P. 89–98 (1998). R.. R. Niemel€. Ando. 23.O.. M. 71–85 (1991).S. and M. Biochem.M. Silva. J.. Technol. Converti. Cruz. 176–180 (1990). G. Moletta. 57–58. 747–759 (2003). Mechanism of formation of 2-furaldehyde from D-xylose. 19. Puls. Curr. Mackie. Aguilera and J. Biotechnol. Yeast. Ethanol production from agricultural biomass substrate. P. W.M. Ingram. 1–14 (1984). 49–56 (1996).. Ethanol fermentation of crude acid hydrolyzate of cellulose using high-level yeast inocula. Department of Agriculture. K. 217. J. Zilli.M. Carbohyd. 141–151 (1999). and G. and L.N. The mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U. Poutanen. The Saccharomyces cerevisiae aldose reductase is implied in the metabolism of methylglyoxal in response to stress conditions.S. Dominguez. 27.R.1 Acknowledgments This study was supported by the National Research Initiative of the USDA Cooperative State Research. T. S. S. 12. Carbohyd... and M. C. Pichia stipitis. Res. J. L. Adv. J. 24/25. Biochem. 1013–1020 (2000). J. Response to acetaldehyde stress in the yeast Saccharomyces cerevisiae involves a strain-dependent regulation of several ALD genes and is mediated by the general stress response pathway. Microbiol. E. Improvement in fermentability of steamed hemicellulose a hydrolysate by ion exclusion. Domiguez. 28–33 (1980). Ohta. Int. J. Mieres. Buchert. Chung and Y. 20.Y. and Candida shehatae.254 Ethanol and Butanol Studies using genomic approaches to understanding inhibitor stress tolerance will allow a better understanding of cell response and in situ detoxification by various fermentative microorganisms.S. 308–315 (1985)..M. Rivista Icidca.T. Education and Extension Service. and J. A. 220–225 (1996). and G. Gong.M.G. References J. Navarro. M. Fermentation inhibitors in wood hydrolysates derived from the softwood Pinus radiate.S. K. 296–303 (1991). Zymomonas mobilis.. Dominguez. Baquinero. Biotechnol.. Beall. R. A. Prata. Felipe.8. and A. Mok. Antal. Characterizacion quimica de eluentes de pulppeoquimeco a la soda de bagazo. K.B. 70–72. Delgenes. Pretreatment of sugar cane bagasse hemicellulose hydrolyzate for xylitol production of Candida guilliermondii. Chem. Debaromyces hansenii and Candida guilliermondii. Clark and K. Wood hydrolysis and hydrolyzates detoxification for subsequent xylitol production. Biotechnol.

Ingram. Biochem. Vibe-Pedersen. Nilvebrant. H. Nilvebrant. 91–104 (1999a). Schmidt. 66. and J. Duff and W. X.. Mol. 55. Ahring. S. 57. 71.S. Microbiol. toxicology and biological implications. Khan and S. B. Biotechnol. Palmqvist. Keweloh. and J. Heipieper. Larsson. Ahring. H. 204–209 (1984). J€nsson.. Biotechnol. Biol.R.A. Appl. Skory.J. J. Biochem. M. and N. and B. Stenberg.. H. 39/40. Gorsich. 121. C. J.. Relative fermentability of lignocellulosic dilute-acid prehydrolysates: application of Pichia stipitis-based toxicity assay. and M.T. M. B. Klinke. and A. 97.. Chem... Ahring. Fernadez. Thomsen. and C. and TKL1 in Saccharomyces cerevisiae.S. Methylglyoxal in living organisms: chemistry. and H. 40. H. Microbiol. Chen. Biochem. Trends Biotechnol. Biochem.. Winkler. S. Appl.B. Qureshi.F. Biotechnol. 49. .A. and B. Biotechnol.. Nichols. 83–88 (1993). N. Comparison of different methods of the o detoxification of lignocellulosic hydrolysates of spruce. A. Z.J. H. 110.B.A. and C.B. E. Biotechnol. and B. J. DeBont. Furfural formation and behavior. Toirkens.B. 1998 L. Thomsen. 5.K. Tolerance to furfuralinduced stress is associated with pentose phosphate pathway genes ZWF1. Thomsen.S. 154–164 (2006). N. J. 145–157 (1999). Bioconversion of forest products industry waste cellulosics to fuel ethanol: a review. Gutierrez. Penner. A.J. 12.B. Appl. Appl. Potential inhibitors from wet oxidation of wheat straw and their effect on growth and ethanol production by Themoanaerobacter mathranii.-O. Klinke. Appl.P. 1460–1469 (2007). Larsen.. Preston.K. Enzyme Microb. J. J€nsson. Biochem.. Lett. Hadi. Blaschek. T. 15–26 (2002). Biores. Preston.N. A. Biosca. 32. Appl. 631–638 (2001). 145–175 (1999). Larroy. Nilvebrant. 96. Ingram. Biotech. Biotechnol. Q.. 163–172 (2002). G. Ind. Reduction of furfural and furfuryl alcohol by ethanologenic strains of bacteria and its effect on ethanol production from xylose. J€rgensen. Palmvist.J. Pretreatment of sugar cane bagasse hemicellulose hydrolysate for ethanol by yeast.H..O.L.W. 151–159 (1999b).D. Biotechnol. C. 409–415 (1994). Biores. a The generation of inhibitors during dilute acid hydrolysis of softwood. Ezeji.L.S. M. Appl.O. Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass. J. M. Bioeng. GND1. Biotechnol.. and J. T. Fenske. K. H. Pares. 98–100. Zacchi. Gutierrez. C.Biomass Conversion Inhibitors and In Situ Detoxification 255 S..F. A. 379–385 (1994).. Gong. C. and J..K.. Dunlop. 24. Mechanisms of resistance of whole cells to toxic organic solvents. Evolutionary engineering of mixed-sugar utilization by a xylose-fermenting Saccharomyces cerevisiae strain. A. and L.B. Chen. Buszko.F. Butanol production from agricultural residues: Impact of degradation products on Clostridium beijerinckii growth and butanol fermentation. A.B. S. Biochem. Klinke. Slininger. van Dijken. Kuyper.J. A. Thomsen. J. 738–747 (2003). Kalapos. E. Liu. Appl.P. 691–697 (1998). Biotechnol..M. Characterization of degradation products from alkaline wet oxidation of wheat straw. Technol. L.P. Tengborg. Liquification of lignocellulose at high-solids o concentration.. N. Diderich.J. 327–340 (2002). Hashimoto. Purification and characterization of a furfural reductase (FFR) from Escherichia coli strain LYO1: An enzyme important in the detoxification of furfural during ethanol production. E. Characterization of the Saccharomyces cerevisiae YMR318C (ADH6) gene product as a broad specificity NADPHdependent alcohol dehydrogenase: relevance in aldehyde reduction. J€nsson et al. B. Sikkema. Felby. 339–349 (2006). 925–934 (2005). Technol. RPE1. biochemistry.J. 10–26 (2004). Int. P.J. L. H.. and J.. Weber. Microbiol. 77. Bioeng. Biotechnol. 361. Ahring. 862–870 (2007). F. Potential inhibitors from wet oxidation of wheat straw and their effect on ethanol production of Saccharomyces cerevisiae: wet oxidation and fermentation by yeast. Hahn-H€gerdal. o o a Detoxification of wood hydrolysates with laccase and peroxidase from the white-rot fungus Trametes versicolor. 82. J.K.A. T. Murray. Reimann. Bioeng. A. L. Klinke. Pronk. Biotechnol. Eng. Olsson. 73. Technol.J. 81.G. Microbiol. and L. and B. M. FEMS Yeast Res. Inactivation and repair of bacteriophage lambda by furfural. Dien. L.B. Hahn-H€gerdal. Larsson.B. Toxicol. Gonzalez. 1–33 (1996). N.

K.N. N. 125–131 (2004). Johnson. Ind. A.L.N. Transcriptome dynamics of ethanologenic yeast in response to 5hydroxymethylfurfural stress related to biomass conversion to ethanol.L. Microbiol.5-bis-hydroxymethylfuran.. Z. Y.S. Microbiol. Liu. and S. Influence of lignocellulosic-derived aromatic compounds on oxygen-limited growth and ethanolic fermentation by Saccharomyces cerevisiae.J. Effects of overexpression of Saccharomyces cerevisiae o Pad1p on resistance to phenylacrylic acids and lignocellulosic hydrolysates under aerobic and oxygen-limited conditions.J. Sinha. and P.W. Ethanol production using concentrated oak wood hydrolysates and methods to detoxify. Liu. Dien.S. Larsson. Biochem.P. J. Isolation of microorganisms for biological detoxification of lignocellulosic hydrolysates. Blanch. Microarray Gene Expression Data Society Meeting. Leonard and G.J. and B. M. Biotechnol. Adaptive response of yeasts to furfural and 5-hydroxymethylfurfural and new chemical evidence for HMF conversion to 2.). Technol.. Shin. M.J. B. 17–36. P. D. 73.W. Microbiol. Collins. S. Liu and P.. Z.J. Nilvebrant. Synthesis. Liu and S. Hajny. C. Biotechnol.. J. Slininger. Biochem. and R. 2005. Biomass Bioenergy. Nilvebrant.J.L. Wall. Z. Kurtzman.J. Mendez-Vilas (ed.L. Duffield (eds). P. C. Germany.J.A. Development of genetically engineered stress tolerant ethanologenic yeasts using integrated functional genomics for effective biomass conversion to ethanol.L. J. Liu.W. J. Appl.. chemistry and applications of 5-hydroxymethylfurfural and its derivatives. Lewkowski. Appl. Enhanced biotransformation of furfural and 5-hydroxymethylfurfural by newly developed ethanologenic yeast strains. Lignocellulosic biomass conversion to ethanol by Saccharomyces.Y. 617–632 (2000). p. A. B. Microbiol. Multiple gene mediated aldehyde reduction is a mechanism of in situ detoxification of furfural and HMF by ethanologenic yeast Saccharomyces cerevisiae. Appl.. 31. 119 (2006). Park. Slininger. C. 27th Symposium on Biotechnology for Fuels and Chemicals. Gorsich.J. Microbiol. Harwood. pp. 81. ARKIVOC.J. and S.O. Appl. 17–54 (2001). Biochem. 65. Identification of potential fermentation inhibitors in conversion of hybrid poplar hydrolyzate to ethanol. Slininger. 77–79.L. Lyer. Eng.J. 64. Transcriptional regulatory analysis reveals PDR3 and GCR1 as regulators of significantly induced genes by 5-hydroxymethylfurfrual stress involved in bioethanol conversion for ethanologenic yeast Saccharomyces cerevisiae. Demain (eds). UK. H. Microbiol. Lee. Z. 1163–1170 (2001a). Wiley-VCH.J. P. Dilute-acid-hydrolysis of lignocellulosic biomass. Biotechnol. J€nsson. J€nsson. in Agriculture as a producer and consumer of energy. 169 (2006). Brink. and A. 386–395 (2003). Lopez. 743–753 (2008b). Lee. J€nsson. 451–460 (2005). 2008a. Luo. 32. DC. Appl. Nichols. Weber. 22. .. N. R. Wallingford. Slininger. J. W. and H. Gorsich. Quintana-Sainz. Liu. Saha. Ind. Dien. Torget. Appl. Enzyme Microb. Genomic adaptation of ethanologenic yeast to biomass conversion inhibitors. 84–86. Fermentation of wood sugars to ethyl alcohol. Slininger. Lee. in Modern Multidisciplinary Applied Microbiology: Exploiting Microbes and Their Interactions. C. Outlaw.S. S.. Adv.256 Ethanol and Butanol S. Liu and P.L.. Berhow. J.O. Comparison of the resistance of industrial and laboratory strains of o Saccharomyces and Zygosaccharomyces to lignocellulose-derived fermentation inhibitors. Appl. Environ. Slininger. B. Bothast. Reimann. and L. Martin and L. P. Biotechnol. Induction of pleiotropic drug resistance gene expression indicates important roles of PDR to cope with furfural and 5-hydroxymethylfurfural stress in ethanologenic yeast. Chang. Z. 390–395 (1945). Chem. 1. Andersh. Biotechnol. Larsson. 345–352 (2004). 37. 125–138 (2002). Washington. Z. Andersh. Liu.S. 93–115 (1999). S. C. Larsson. and S. P. and L... N. Z. 2006.C.C. B. J. 67. Z. P.L.J. Moreno. 167–174 (2001b). Development of a Saccharomyces cerevisiae strain with o enhanced resistance to phenolic fermentation inhibitors in lignocellulosic hydrolysates by heterologous expression of laccase.. Biochem.G.K. 547–559 (1999). ASM Press. 9. in Bioenergy. and R.L.H. 57. Biotechnol. 27–36 (2006). Biotechnol. Eng. J. CAB International. and L. 121–124. Biotechnol. and Y. Biotechnol. A.J. Moon. Chang. Appl. Cassland. Liu. Z.J. Slininger.

J. C. 185–197 (1997a). Technol. McMillian. Dominguez. Petersson.L.M. Appl. Galbe. Rakhmilevich.D. Biochem. A. N.. J. T. Biotechnol. Lee. Elander.J. Detoxification of lignocellulose o hydrolysates with ion exchange resins. Gidroliz. Hahn-Hagerdal. Biores. and L. Liden. M. 67. Prog. 98. and G. Improved xylitol production with Debaryomyces hansenii Y-7426 from raw or detoxified wood hydrolysate. B. Palmqvist and B. a A 5-hydroxymethylfurfural reducing enzyme encoded by the Saccharomyces cerevisiae ADH6 gene conveys HMF tolerance. Guisado. and J. J.. Hahn-H€gerdal. Himmel. Technol.C. 93. 19. 16–17 (1991).. and M. 455–464 (2006). Transformation of yeast growth inhibitors which occurs during biochemical processing of wood hydrolysates. Washington.Biomass Conversion Inhibitors and In Situ Detoxification 257 C. Almazan..I. 91–93. Enzyme Microb. and L. Ranatunga.. Wells.. L. J. 363.E. 470–476 (1996).. and R. Zacchi. Technol. Biotechnol. Nilsson. and M.. Parajo. Biochem. Jervis.C.Y. J.. and M.J. Palmqvist. Dominguez. A. 5. J.. 286–293 (1997). Technol. Modig. Gusarova. 17. K. Reimann. Technol. M. Technol.. 18. Biochem. Almeida. 7866–7871 (2005). M. Biores. Biotechnol. Hahn-H€gerdal. 379–390 (2005). M. Baker. Simultaneous detoxification a and enzyme production of hemicellulose hydrolysates obtained after steam pretreatment. Nemirovskii and V. Gorwa-Grauslund. Lesokhimm. 1767–1773 (2007). York.. Appl. Enzyme Microb. Biores. Palmqvist. in Enzymatic Conversion of Biomass for Fuel Production.F. Influence of furfural on anaerobic glycolytic a kinetics of Saccharomyces cerevisiae in batch culture. Bioeng. Ingram. Larsson. Morimoto and M. and K. Lopez. B. N. F. R. Alternatives for detoxification of dilute-acid lignocellulosic hydrolyzates for use in fermentative processes: a review.C. 442–446 (1967). Bioabatement to remove inhibitors from biomass-derived sugar hydrolysates. Biotechnol. and L. Murakami. and B. . H. Sizov and V. Technol.D.N. Prom-st. G. M. 18–24 (1997). S. J. Yeast. 17–24 (2000a). Dominguez. J€nsson. and B. Appl. Inhibition effects of furfural on alcohol dehydrogenase. T. 62. Nilvebrant. Fermentation of lignocellulosic hydrolyzates. B. Marcet. V. Almeida. G.P. Dominguez. Hahn-Hagerbal. McMillan. Y. J. 1–10 (2004). Hatzis. 769–776 (2002). G. Gorwa-Grauslund. E. Martin. Biotekhnologiya. J.. Helm.. A. and M. Z.E. Detoxification of dilute acid hydrolysates of lignocellulose with lime. Technol. Holtzapple. Modig.O. 673–686 (2005). J. B. DC. 124. A. S.. The effects of water-soluble inhibitors a from steam-pretreated willow on enzymatic hydrolysis and ethanol fermentation. Enzyme Microb. Nemirovskii. Moisier. 1. Hahn-H€gerdal. O. 21. E. Y. and G. Pathways of furfurol and oxymethyl furfurol conversion in the process of fodder yeast cultivation. Kostenko.J. T. Microbiol. B.. 1994. Technol. Reczey. E. Adaptation of a recombinant xylose-utilizing o Saccharomyces cerevisiae strain to a sugarcane bagasse hydrolysate with high content of fermentation inhibitors. N. B. M.O. 66. H. and J. Dien. 35–49 (2001). Dale. Features of promising technologies for pretreatment of lignocellulosic biomass. Part 3: Operation in culture media made from lignocellulose hydrolysates. Technol. Lindn. R.. V. L. Enzyme Microb. Biores. and C. 312–331 (1996). Hahn-H€gerdal. Olsson. 45. Fermentation of lignocellulosic hydrolysates for ethanol production. Kostenko. 25–40 (1998). S. Biores.W. Overend (eds). Mussatto and I. Roberto. Biores. 285–289 (1989). American Chemical Society. Ladisch.. Technol. 96. Environ. Cofactor dependence in furan reduction by Saccharomyces cerevisiae in fermentation of acid-hydrolyzed lignocellulose.D. 23.S. Appl.F. Rodriguez. Taherzadeh. Pretreatment of lignocellulosic biomass. Biochem. Szengyel. J. Parajo. 287–293 (2001). Karhumma. 71. Hahn-H€gerdal. e aldehyde dehydrogenase and pyruvate dehydrogenase.O.M. 25–33 (2000b). Martinez. E. Studies on fermentation products from aldehyde by microorganisms: the fermentative production of furfural alcohol from furfural by yeasts (part I). Biotechnol. Zacchi. Biotechnological production of xylitol. E. Ferm. M. A. Nichols. 74. Fermentation of lignocellulosic hydrolysates II: inhibitors and a mechanisms of inhibition.F. J€nsson. S. Wymann. 20. Identification of inhibitory components toxic toward Zymomonas mobilis CP4(pZB5) xylose fermentation.R.J. Hahn-Hagerdal. 447–454 (1999). Palmqvist and B. I. 74.. Preston.M. Palmqvist. Inhibition and detoxification.

Improvement of xylitol production by Candida guilliermondii FTI 20037 previously adapted to rice straw hemicellulosic hydrolysate. and I.J. L.H. Effects of furfural on e respiratory metabolism of Saccharomyces cerevisiae in glucose-limited chemostats. Taherzadeh. Taherzadeh. Lett. Rodrigues. M. Microbiol. Conversion of furfural in aerobic and e anaerobic batch fermentation of glucose by Saccharomyces cerevisiae.C. Gomez. A linear discrete dynamic system model for temporal gene interaction and regulatory network influence in response to bioethanol conversion inhibitor HMF for ethanologenic yeast. 73. 2349–2353 (2007). Effects of furfural and 5-hydroxymethylfurfrual on the fermentation of Saccharomyces cerevisiae and biomass production from Candida guilliermondii. Ethanol production from glucose and dilute-acid hydrolysates by encapsulated Saccharomyces cerevisiae. W. Chem. . Lourenco. Technol. Silva.. Cheng. 69. Felipe. Tsuchida.S. Technol. Lettinga. J. 345–353 (2005). Sarvari Horvath. 18. O.258 Ethanol and Butanol T. T. 77–95 (2007). 213. Gustafsson.C. Monteiro. Biotechnol. and M. C.B.V. Tech.L.S.J. Y. Okino.D...M.C. M. Roberto. 169–174 (1999). Y. Silva. Dickinson. M. C. 50.C.S. 19. and S.G. 90. L. Appl.. Silva and I. Niklasson. M.. J. Biores. I. Silva and I. 164. F. Niklasson. Microbiol. F. Appl. L. Microbiol. Lindn. Utilization of sugar cane bagasse hemicellulosic hydrolysate by Candida guilliermondii for xylitol production. Helm. L. Microbiol. R.G. C. Microbiol. Biotechnol. The influence of pH.J.S. Inui. and S. Environ. Hemicellulose bioconversion. Lett. A. Midgley. Identification and disruption of the gene encoding the K( þ )-activated acetaldehyde dehydrogenase of Saccharomyces cerevisiae... Sanchez and J. Silva.A. McMillian. Appl.. 738–743 (2001). Sakai. Lecture Notes Bioinfomatics. Biotechnol. Hatzis. Appl. Watanabe.. B. Sierra Alvarez and G. Niklasson. Biotechnol. Tessier. M. 4076–4086 (2003). Felipe. Lindn. Effect of lignocellulose-derived inhibitors on growth of and ethanol production by growth-arrested Corynebacterium glutamicum R.. 36. P.S. J. Talebnia. Chem. Ferment. Jervis. M. Food Energ.. Mancilha. Xylitol production by Candida guilliermondii FTI 20037 grown in pretreated sugar cane bagasse hydrolysate. 10. Ind. Statistical screening method for selection of important variables on xylitol biosynthesis from rice straw hydrolysate by Candida guilliermondii FTI 20037.. Ferreira-Dias. Ribeiro. P. Microbiol. 1–11 (2002). Sene.J. Bautista.G. Taherzadeh. and G. Almeida e Silva. 29–34 (1998). Roberto..A.J. J. Yukawa. M. Eur. 57.A. Environ. Gustafsson. Vitolo. Sauer. and C. Technol. J.. Liu. Lett. S. Appl.. Microbiol. 1990–1998 (2003). Bioeng. 32. 299–311 (2001). Saha. Appl. 53. Sustain Agr. and G.J. Hydrolysis of lignocellulosic materials for ethanol production: a review.G. Technol. and M. Ichihashi. S. 83. Lacis. Food Res. 315–318 (1988). M. 743–747 (1999). Felipe. FEMS Microbiol. Braz. 248–252 (2001). The methanogenic toxicity of wastewater lignins and lignin related compounds. Sun and J.D. Evolutionary engineering of Saccharomyces cerevisiae for anaerobic growth on xylose. Taherzadeh. Toxicity of hard-wood extractives toward Saccharomyces cerevisiae glucose fermentation. Felipe. 271–275 (1991). 32. 1116–1119 (1998). Metabolic study of the adaptation of the yeast Candida guilliermondii to sugar cane bagasse hydrolysate. C. C.J. Physiological effects of 5-hydroe xymethylfurfural on Saccharomyces cerevisiae.M. Franzen. Environ. Vitolo. and P. 443–455 (1991). Sonderegger and U. Roberto.B. Converti. M. Zilli.D.C. 132–138 (2001). I.. H. Biores.S. B.A. Ranatunga. R. Ind. J. 30. Enzyme Microb. S. 1125–1127 (1997b).A. Meaden. and H.L. J.G. Lindn.M. Eng. Song and Z. and G. M. R. M. 279–291 (2003).L.F. Biotechnol. 69. Kawaguchi. C. Niklasson.M. M. C.P. 701–708 (2000). J. Biotechnol. and M. Bioeng..C. 4532. 87. temperature and hydrolysate concentration on the removal of volatile and nonvolatile compounds from sugarcane bagasse hemicellulosic hydrolysate treated with activated charcoal before or after vacuum evaporation. S. M. Kinetics of selective absorption of impurities from a crude vegetable oil in hexane to activated earths and carbons.. Technol. Biotechnol..

and L. 67. Appl. 5668–5674 (2001). Putative xylose and arabinose reductases in a o a Saccharomyces cerevisiae. 5-hydroxymethylfurfural. Hahn-H€gerdal. Microbiol. Biotechnol. J. Environ. Wyman.Biomass Conversion Inhibitors and In Situ Detoxification 259 K.. 68. Enrique. Yeast.E. Effects of alcohol compounds found in hemicellulose hydrolysates on growth and fermentation of ethanologenic Escherichia coli. Deletion of the GRE3 aldose a a reductase gene and its influence on xylose metabolism in recombinant strains of Saccharomyces cerevisiae expression of the xylA and XKSI genes. C.P. Chambers.. Guerra. Biotechnol. M. Bartroli. E.V. M. Bioeng.3-butanediol fermentation of mannose-rich prehydrolysates. Martinez. Penas.. 1233–1241 (2002).F. Bioeng. R.Y.O. Iglesias. Bioeng. and L.P. Biotechnol. Elander.P. M.J. 19. M. Biores. C. 23. B.. D. Lignin and extractives derived inhibitors in the 2. and B.L. 68. R. L. 203–210 (1999). Hahn-H€gerdal. and B. J€nsson. R.O. G. A. Redondo. A. 7. 509–512 (1992). 172–178 (2002). Lett. Chambers. Appl. A.E. 78. 12. Effects of organic acids on growth and fermentation of ethanologenic Escherichia coli LYO1. 524–530 (1999). Villa. Wahbom and B. Hahn-H€gerdal.L. Comparative sugar recovery data from laboratory scale application of leading pretreatment technologies to corn stover.V. Ladisch and Y. Zaldivar. Biotechnol. Holtzapple. Acta Biotechnol.R. 96...O. Microbiol. J. 191–197 (1986). Furfural. and M. Microbial transformation of furfural to furfuryl alcohol by Saccharomyces cerevisiae. Lee.H. Tr€ff. Tran and R. Bioeng. Tran and R. 524–530 (2000). Lopez. Technol.. Diaz. Martinez. Tr€ff. Rodriquez. Red oak wood derived inhibitors in the ethanol fermentation of xylose by Pichia stipitis CBS 5776.. W. Ingram. and acetone act as external a electron acceptors during anaerobic fermentation of xylose in recombinant Saccharomyces cerevisiae. Effects of selected aldehydes on growth and fermentation of ethanologenic Escherichia coli. 841–846 (1985).R. Biotechnol. I. van Zyl. Dale. Zaldivar. Otero Cordero. Ingram. R. K. 2026–2032 (2005). Zaldivar and L. A. J.T. Biotechnol. Ingram. 66.. M. .


. HahnH€gerdal et al. and hemicellulose is a branched polymer which. 2003.1). may also contain the hexose sugars mannose and galactose. Hans Blaschek ` ..1. 2005). 2007a.. 1993). Jeffries.. and hemicellulose (Figure 13. a a Biomass to Biofuels: Strategies for Global Industries and Hideaki Yukawa Ó 2010 John Wiley & Sons.. Technical and economical assessments estimated that the complete conversion of pentose sugars to ethanol would reduce the production cost of bioethanol by as much as 22 % (Sassner et al. cellulose is a linear polymer of glucose.. In such raw materials pentose sugars can make up more than 35 % of the total dry matter (Hayn et al. Lignin is a heterogeneous polymer of substituted aromatic building blocks. Therefore. and the pentose sugars xylose and arabinose (Lynd et al. cellulose.1 Introduction Lignocellulosic Raw Material Lignocellulosic raw material primarily consists of lignin. Ltd Edited by Alain Vertes.13 Fuel Ethanol Production From Lignocellulosic Raw Materials Using Recombinant Yeasts Grant Stanley and B€rbel Hahn-H€gerdal a a 13. agricultural residues and grasses. 2007b). 2008). Pentose sugars are primarily present in hemicellulose derived from hard woods. Nasib Qureshi. 2006. in addition to glucose. considerable research investments have been made over the past three decades to construct and develop microorganisms that efficiently ferment both hexose and pentose sugars to ethanol (Dien et al.1 13. H€gerdal et al.

1 Composition of lignocellulosic raw material and its hydrolysis and degradation products .262 Extractives (1-5%) Lignin (21-32%) Ash (0-2%) Hemicellulose (19-34%) Cellulose(33-51%) HO O OH OH HO OH Ethanol and Butanol CH2OH O OH OH OH Galactose HO OH CH2OH O OH CH2OH O OH OH Xylose Glucose OH OH Sugars O HO HOH2C OH Mannose OH OH HO O OH CH2 OH OH Arabinose Rhamnose R O CHO R R OH CH 2 O HO CHO Furans Furfural O Phenolics HMF Inhibitors O O OH H3C OH Weak acids OH O Wood resin Acetic acid Formic acid Levulinic acid Figure 13.

.1. 1997). First. 13. 1983. engineering of the product formation metabolism of industrial strains to improve ethanol yield is a prerequisite for attaining optimal process economics. various species of bacteria. During the pretreatment and acid hydrolysis steps.1.. 1990). Jeffries. alkali or organic solvents (Galbe and Zacchi.. 2007) to render the carbohydrate fractions accessible to hydrolysis by either acids or enzymes. 1996. 2003). When combined with the high ethanol sensitivity of a number of recombinant microorganisms that have been created for the lignocellulose-to-ethanol industry (e.2 Fermenting Microorganisms In Nature. 2007a). 2004.. Skoog and HahnH€gerdal.3. 2005). SSF indicates simultaneous saccharification and fermentation when hydrolysis is performed with enzymes The conversion of lignocellulosic raw materials to fermentable sugars involves a sequence of operational steps (Figure 13. and furans and weak acids are formed from solubilized hexose and pentose sugars (Figure 13. 1993. the benefits of improving the ethanol tolerance of ethanologenic production strains thus become clear. .. which adds to the production cost of ethanol. it is predicted that process improvements in the future will enable considerably higher sugar yields... Hahn-H€gerdal et al.2). 1987. the major impacts of which are lower fermentation rates.. microbial sensitivity to the toxicity of ethanol can affect fermentation productivity.2 Schematic representation of the conversion of lignocellulosic raw material to ethanol.. Bothast et al. Ingram et al.Fuel Ethanol Production From Lignocellulosic Raw Materials Using Recombinant Yeasts SHF Lignocellulose Pretreatment Solubilization of Biomass hemicellulose Hydrolysis Cellulose hydrolysis using Acid / Enzymes 263 Fermentation Conversion of sugars to ethanol by yeast Distillation Ethanol SSF Figure 13. multiple molecular events occur: acetic acid is released from hemicellulose. Toivola et al. reduced ethanol yields and decreased microbial lifespan (Stanley et al.. 1999. however. Although the relatively low sugar concentrations that typically characterize the currently produced lignocellulose hydrolysates do not result in final ethanol concentrations that are high enough to significantly affect fermentation productivity. Escherichia coli KO11. This has been most successfully accomplished for the bacterium E. 1984. 2007). A similar optimization of obligatory anaerobic bacteria is currently under way (Tyurin et al. Most fermenting microorganisms require that the lignocellulose hydrolysate is detoxified prior to fermentation (Hahn-H€gerdal and a Pamment. a Alcoholic fermentations are also constrained by the toxicity of the end product on the production strain. phenolic compounds are solubilized from the lignin fraction. Almeida et al. the raw material is pretreated at elevated temperatures in the presence of acids. 1987).g. a Consequently.. Stanley et al.. Specifically. Dien et al. filamentous fungi and yeasts ferment pentose sugars to ethanol (Figure 13. Hespell et al. coli (Ingram et al. most of these organisms also form an array of byproducts.

3. neither organism can ferment xylose or arabinose. There is an obvious economic advantage in using a single microorganism to perform all of these functions in lignocellulose-based ethanol production. It has also been the goal of some research groups to simplify the lignocellulose-to-ethanol process by combining lignocellulose hydrolysis and fermentation to ethanol into a single operation. Namely. mobilis has been elegantly engineered for xylose and arabinose cofermentation to ethanol (Zhang et al.. coli Filamentous fungi Pentoses P. stipitis Ethanol Z. 2008). the dashed lines indicate pathways requiring metabolic engineering .. the naturally xylose-fermenting yeast Pichia stipitis produces ethanol from xylose with stoichiometric yields (Figure 13. However. Boxma et al. 1990).3... Z. 2004). However. 2006). while the bacterium Zymomonas mobilis and the baker’s yeast Saccharomyces cerevisiae both ferment hexose sugars to ethanol with close to theoretical yields (Figure 13. The so-called manufacturing approach of Consolidated Bioprocessing (CBP). Therefore. 1986. For example. The solid lines indicate pathways already present and functional. species of filamentous fungi ferment pentose sugars to ethanol in addition to other products (Figure 13. On the other hand. Wu et al. this is achieved only under conditions of a very low and extremely well controlled levels of oxygenation (Rudolf et al. CBP technology potentially requires lower capital. describes the consolidation of cellulase production.264 Ethanol and Butanol Similar to bacteria. Skoog and Hahn-H€gerdal. cerevisiae Figure 13.3 Microorganisms considered for pentose fermentation.3).. 2005). materials and utilities costs compared to dedicated cellulase production and fermentation systems.. cellulose hydrolysis and sugar fermentation steps into a single unit operation (Lynd et al. metabolic engineering strategies are also being applied to these organisms to redirect product formation (Panagiotou et al.. with some estimates placing CBP costs at 25 % of the cost for simultaneous saccharification and cofermentation Anaerobic lignocellulose fermenting bacteria E. with reference to the lignocellulosic ethanol industry. 1995. Deanda et al. mobilis S. 1996).

cerevisiae are addressed later in this chapter. 2005). although bacterial cellulases have also been successfully produced (e. 13. cellobiose and xylan...Fuel Ethanol Production From Lignocellulosic Raw Materials Using Recombinant Yeasts 265 with dedicated cellulase production. a a The construction of an S. Hahn-H€gerdal et al. cerevisiae to date are of fungal origin..g. in addition to having the ability to concurrently ferment both hexose and pentose sugars to ethanol. have not yet been reported.. cerevisiae There are numerous reports on the expression of cellulases in S. or discovering. Most of the cellulases that have been expressed in S. may lend themselves more to CBP technology.2 Consolidated Bioprocessing and Ethanol Production The significant challenge in developing commercial CBP lies in constructing. Cho et al. 2007a. The strain is currently being investigated for commercialization using CBP-based technology (Ebert. it is an organism generally recognized as safe (GRAS) and its genetics and physiology are well defined (Hahn-H€gerdal et al.. since the recombinant strain must be able to produce and secrete heterologous saccharolytic enzymes at high levels. fuelling speculation on whether such an organism could exist. either naturally or by design. CBP. However. cerevisiae strains for such processes. a process organism that can perform the task. a unique bacterium of the genus Clostridium (designated Clostridium phytofermentans) was isolated from forest soils. this microbe is able not only to hydrolyze cellulose. (ii) exoglucanases. including its ethanol yield. 2007). In particular. van Zyl et al. no native organism had been found with all of the necessary traits. Chung et al. and (iii) b-glucosidases (Table 13. 1997. 2007b).. The attraction of using S. and Aspergillus spp. Interestingly. the aim of this chapter is to describe the progress that has been made to date in developing yeast strains that will simplify and increase the productivity of the lignocellulose-to-ethanol process. cerevisiae for CBP is its industrial robustness.. Until recently. with their broad substrate range and product diversity. mainly from Trichoderma (recently renamed Hypocrea) spp. With the above in mind. but also to ferment a range of hexose and pentose sugars to a mixture of organic acids and ethanol (Warnick et al. productivity and tolerance. 2002). which includes its high stress tolerance. pentose fermentation and ethanol inhibition are discussed. consequently improving process economics. in addition. 13. Although many bacteria and fungi. details on the growth and fermentation characteristics of C. A potential problem with . 1999). the focus of the next section is on conferring to S.1. and 50 % of the cost for the entire process (Lynd et al. covering the three main areas of activity necessary for complete cellulose hydrolysis: (i) endoglucanases. Recently. from Clostridium spp.1 Cellulase Expression in S. cerevisiae the ability to produce extracellular saccharolytic enzymes with sufficient activity to support growth and fermentation on cellulose. high ethanol productivities and yield. however. The challenges and achievements in developing a pentose sugar-utilizing S. phytofermentans. there is considerable interest in modifying S.. 2007)... cerevisiae strain that can directly ferment cellulose to ethanol is a sizeable task. and Bacillus spp. cerevisiae.2.

(1988) a Reinikainen et al. (1998) Hong et al. (1988) van Rensburg et al. MUL BMCC. PASC PNPC b-glucan PNPG. cerevisiae Organism source and enzyme (gene) CELLULASES Cellobiohydrolase: Aspergillus aculeatus CBHI Aspergillus niger CBHB Cellulomonas fimi Exg Phanerochaete chrysosporium CBH1-4 Specific activity Substrate (U mgÀ1) Reference 0. Lichenan. MUC b-glucan CMC. (1987) a Saloheimo et al. CMC. (1998) den Haan et al.007 — 3. (1998) den Haan et al. (1997) Hong et al.22 (PASC) — — — — 204 — — — 107. b-glucan. Lichenan. (1995) van Arsdell et al..1 Cellulase and hemicellulase expression by S. (2002b) Penttil€ et al. (1997) Saloheimo et al. CMC. (2007) Wong et al.3. (2003) Xie et al.1. (2001) Cho et al. (1996) van Rensburg et al. (1988) a Fujita et al. (2007) Penttil€ et al. (1987) Penttil€ et al.26 (BMCC) 0. MUL. PASC CMC CMC CMC b-glucan CMC Avicel b-glucan CMC CMC. PASC Amorphous cellulose BMCC.4glucanase Clostridium thermocellum (celA) Thermoascus aurantiacus EGI Trichoderma longibrachiatum EG Trichoderma reesei EGI EGII EGIII EGIV EGV — — — — . b-glucan b-glucan Takada et al. (1992) den Haan et al.266 Ethanol and Butanol Table 13. PASC Amorphous cellulose PASC BMCC. (1994) Trichoderma reesei CBHI — — 0. (2007) Penttil€ et al. (2004b) den Haan et al. b-glucan.1 — 15 — CBHII Endoglucanase: Aspergillus aculeatus CMCase Aspergillus niger (eng1) Bacillus circulans Endo/Exo bifunctional enzyme Bacillus subtilis Endo-b-1. (1999) Hinchliffe and Box (1984) Chung et al. (2007) Murai et al. PASC BMCC. (1987) a Fujita et al.6 — — Avicel BMCC.

(1995) Li and Ljungdahl (1996) Moreau et al. APEU Crous et al. 67. Machida et al. (2006) Birchwood xylan Birchwood xylan Xylan Birchwood xylan Birchwood xylan Crous et al. Machida et al.4 — PNPG.0 ATRU 135 ATEU 126 APEU — 2000 — — — PNPA PNPA PNPA ABIU. Gentiobiose. 26.2. (1998) Penttila et al.1. (1988) Cellotriose. ATEU. (2002) Adam et al. (1996) Sanchez-Torres et al. Cellobiose.5. 20. (1992) La Grange et al. 52. (1998) van Rensburg et al. (1996) La Grange et al.1 (Continued) Organism source and enzyme (gene) b-glucosidase: Aspergillus aculeatus BGLI Aspergillus niger BGL Bacillus circulans BGL Bacillus polymyxa (bglA) Endomyces fibuliger BGLI Ruminococcus flavefaciens CEL1 Saccharomycopsis fibuligera BGLI Specific activity Substrate (U mgÀ1) — 25 — — — — — b-glucan Cellooligosaccharides BCIG PNPG Cellobiose Cellobiose PNPC Reference 267 Fujita et al. 27. ATRU.78 Trichoderma reesei (abf1) a-Glucuronidase Aureobasidium pullulans (aguA) b-Xylanase Aspergillus kawachii (xynC) Aureobasidium pullulans (xynA) Cryptococcus albidus XLN Trichoderma reesei (xyn2) — 14.3. Cellotetraose. (1995) van Rensburg et al.7 BGLII 168.1 ABIU 60. (1984) Pack et al. (1996) de Wet et al.Fuel Ethanol Production From Lignocellulosic Raw Materials Using Recombinant Yeasts Table 13. 25. (1998) 43. (1996) Magolles-Clark et al. (1988) Methyl-b-glucoside (activity in same order as substrates) PNPG van Rooyen et al. (2002b) Takada et al. (2001) (continued) . (2005) HEMICELLULASES Xylan degradation: a-L-Arabinofuranosidase Aspergillus niger (abfB) — 5. Gentiobiose (activity in same order as substrates) PNPG.1.

PNPbX ¼ p-nitrophenyl-b-D-xylopyranoside. PNPbG. PGGM PNPaGal. 2005). LBG ¼ locust bean gum. and suggests that protein hyperglycolysation by S. van Rooyen et al. a Reinikainen et al. 1992). PGGM INM.. MUL ¼ methylumbelliferyl lactoside. PNPC ¼ p-nitrophenyl-b-D-cellobioside. PNPA ¼ p-nitrophenyl-a-L-arabinofuranoside. Most reports to date on heterologous cellobiohydrolase production by yeast report a relatively low titer of secreted cellulase (in the range 0. LBG. LBG. which need to be produced and secreted with high titer and activity. 2005. recent evidence has suggested that the . (1996) — — — PNPaGal. cerevisiae strains producing these enzymes with relatively high specific activities (van Arsdell et al. McBride et al. LBG LBG LBG Magolles-Clark et al. 2005). INM ¼ ivory nut mannan. a recent study using a novel approach to measure cellobiohydrolase specific activity.1 (Continued) Organism source and enzyme (gene) b-Xylosidase Aspergillus oryzae (xylA) Trichoderma reesei (bxl1) Mannan degradation: a-Galactosidase Trichoderma reesei (agl1) (agl2) (agl3) Specific activity Substrate (U mgÀ1) — — PNPbX PNPbX. MUC ¼ methylumbelliferyl cellobioside. PGGM PNPaGal. ATRU ¼ aldotriouronic acid. xylobiose Reference Katahira et al.. However. PNPA. PGGM ¼ pinewood galactoglucomannan. (1996) Magolles-Clark et al.. 1987. PNPG ¼ p-nitrophenyl-b-D-glucoside. CMC ¼ carboxymethylcellulose.5% of total cell protein). (2005)  Stalbrand et al. LBG. APEU ¼ aldopentaouronic acid. demonstrated that the glycosylation of heterologous cellobiohydrolase does not significantly affect its specific activity on amorphous cellulose. BCIG ¼ 5-bromo-4-chloro-3-indolyl-b-Dglucopyranoside. This suggests that cellobiohydrolase production is a limiting factor for the successful design of amorphous cellulose-hydrolyzing yeast (Lynd et al. PNPaGal ¼ p-nitrophenyl-a-D-galactopyranoside. 1988. hyperglycolysation does not appear to be a limiting factor for endoglucanase or b-glucosidase enzymes. with reports of S.. cerevisiae may not be a significant issue (den Haan et al. (1996) Magolles-Clark et al. melibiose.002–1. ATEU ¼ aldotetraouronic acid. On a more promising note. The critical components for successful CBP are the cellobiohydrolases. (2004) Magolles-Clark et al. BMCC ¼ bacterial microcrystalline cellulose. (1996) Setati et al. PNPbG ¼ p-nitrophenyl-b-D-glucopyranoside.. (2001) Ximenes et al. Furthermore.268 Ethanol and Butanol Table 13.. cerevisiae is that of enzyme hyperglycosylation. Raffinose. 2007a). ABIU ¼ aldobiouronic acid. which has been proposed to account for observed reductions in saccharolytic activity and the ability of saccharolytic enzymes to bind to crystalline cellulose (Pentill€ et al. xylan. heterologous protein production by S. (1995) b-Mannanase Aspergillus aculeatus — (man1) Orpinomyces strain PC2 179 Trichoderma reesei — (man1) a — ¼ Not reported..

Li and Ljungdahl. 1997). La Grange et al. 2007). significant production of xylose from birchwood xylan can only be obtained when these enzymes are coproduced and secreted (La Grange et al. 1996.. La Grange et al. a recombinant S. 2005. 13. cerevisiae. mainly originating from Aspergillus spp. Likewise.. 2002a). A lead to improving this process is thus to further optimize heterologous enzyme expression in order to increase the amount of glucose released from the cellulose (27 % of the glucose in the PASC). cerevisiae can be anticipated to occur in the near term by focusing research efforts on increasing the expression levels of cellulase systems.. 2004b. reesei. 4-glucanase. cerevisiae strain coexpressing a T. 2007. high levels of b-xylanase and b-xylosidase activity were produced by S. 2001. cerevisiae (i. 2000.1. Kim et al. cerevisiae under the transcriptional control of glycolytic promoters. Sampedro et al. den Haan et al. In particular... endoglucanase II and cellobiohydrolase II (both from T. 2007b.and mannan-degrading enzymes (Table 13. 2001.. 1992. cerevisiae strain has been constructed that not only secretes biologically active endo-b-1. Nonetheless. In all these cases.. Setati et al. however. with the functionality of these constructs verified by the detection of active secreted enzyme (Moreau et al.) More recently. 1996.2.2 Hemicellulase Expression in S. (Note: high cell densities are required for this fermentation as the strain cannot grow on PASC as a sole carbon source.9 g lÀ1 from 10 g lÀ1 phosphoric acid-swollen cellulose (PASC) over 40 h (Fujita et al.. La Grange et al. 2005.. and Trichoderma spp. cellodextrinase and cellobiase.... van Zyl et al. encoding xylan. Park et al. these levels remain well within the reported range of heterologous protein production by S. recently renamed Hypocrea jecorina). Luttig et al.. a S. van Zyl et al. and that is capable of producing ethanol concentrations up to 2.. Fujita et al. cerevisiae can be achieved by constructing expression cassettes of heterologous genes.e. 2004. Notably... the process economics would be improved by using yeast strains with the ability to hydrolyze and ferment any remaining hemicellulosic materials. Multiple cellulase enzymes have been successfully expressed in yeast systems. reesei . cerevisiae strain was constructed that coexpresses b-glucosidase I (from Aspergillus aculeatus). Despite xylan-hydrolyzing enzymes b-xylanase and b-xylosidase having been individually expressed in S. 2002b). 2001). 1–10 % of total cell protein) (Lynd et al. 2007b demonstrated growth on 10 g lÀ1 PASC and subsequent production of 1 g lÀ1 ethanol by a recombinant S.. 2000.. cerevisiae The hemicellulose fraction of lignocellulose is hydrolyzed to varying degrees by most pretreatment methods. but is also capable of both growing on cellobiose and hydrolyzing cellulosic substrates (van Rensburg et al. 1996. cellobiohydrolase. cerevisiae using genes originating from T. A number of studies have been reported which promote the view that the expression of hemicellulases in S. de Wet et al. Fujita et al.. Crous et al.Fuel Ethanol Production From Lignocellulosic Raw Materials Using Recombinant Yeasts 269 cellobiohydrolase expression levels needed to enable growth on crystalline cellulose are achievable for a broad range of cellulase specific activities. this strain represents significant progress toward realization of the CBP process using S.. and as a result to increase the final ethanol titer. As a result. Katahira et al. 1998). significant advances in the performance of cellulose-utilizing S... den Haan et al. 1995.. 2006. reesei endoglucanase and a Saccharomycopsis fibuligera b-glucosidase.

g. the resulting strain produced 7. the hydrolysis from galacto(gluco)mannan of raffinose..g.. cerevisiae to be used for CBP. or a cocktail of saccharolytic enzymes with similar function. (2004) brought this process a step closer to CBP application by introducing genes coding for xylose utilization enzymes (namely: xylose reductase and xylitol dehydrogenase from Pichia stipitis) into a recombinant S. Research on CBP yeast development also needs to identify the optimal combination and activity levels of saccharolytic enzymes to achieve optimum hydrolytic . cerevisiae strains have been constructed that express and secrete endomannanases from A. 2001.. 1996a). cerevisiae strain expressing a-galactosidases from T.3 g gÀ1 xylan. Nevertheless. 1996b. Although in isolation these results have little practical significance regarding complete hemicellulose hydrolysis in CBP.. 2005). 1996. Observations from numerous studies on hemicellulase expression in S. In this regard. Notably. including: (i) increasing the amount of enzyme expressed and secreted by the cell. cerevisiae resulting in measurable enzyme activities (de Wet et al. respectively.. from A. Ximenes et al. oryzae). traditional mutation approaches should be used in conjunction with rational design in the further development of CBP yeast strains. Mannans are the major hemicellulose component of softwoods. From an energetic perspective. Although only 30 % of the reducing sugars present in the xylan could be released before reaching a plateau in ethanol production.. reesei) have also been expressed in S. Today. niger and T.. xylan debranching enymes such as a-D-glucuronidases (e. 1995. Building on this observation. Katahira et al. or (iii) increasing the enzyme specific activity. cerevisiae promote the view that the display and activity of either a single enzyme. In this respect. S. T. a considerable amount of work is still required for achieving the engineering of recombinant yeast strains that alone are capable of growing on. a CBP approach for ethanol production from biomass is closer to becoming a reality. reesei) and b-xylosidase (from A. melibiose and p-nitrophenyl-a-D-galactopyranoside to galactose has been achieved using an S.1 g lÀ1 ethanol over a 62 h period from birchwood containing 0.. the latter is more favorable for a recombinant S. cerevisiae coexpressing xylanase II (from T.. reesei (Margolles-Clark et al. given the recent advances made in constructing S. aculeatus. The design of recombinant yeast strains capable of efficient lignocellulose hydrolysis will need to include ways of increasing enzyme activity toward its substrate. lignocellulose substrates in a single unit operation. reesei and Orpinomyces spp. Sanchez-Torres et al. To take advantage of this fact. it has been speculated that increased xylan saccharification could be achieved by improving enzyme activity and/or codisplaying other xylanolytic enzymes. and producing ethanol from. given that the industrial organism used for manufacturing must support the expression of multiple foreign genes as well as achieve reasonable growth and fermentation rates. (ii) optimizing the ratio of different enzyme activities. This may be achieved by a variety of approaches. cerevisiae strains that coproduce saccharolytic enzymes and are capable of utilizing pentose and hexose sugars to produce ethanol.. from Aureobasidium pullulans) and a-L-arabinofuranosidases (e. 2006. Stalbrand et al. 1996).270 Ethanol and Butanol and Aspergillus spp. is possible.. it is the integration of all the knowledge gained thus far that makes a valuable contribution to the design and construction of an S. all of these strains have been observed to  have activity toward mannan (Setati et al. Notably. this particular experiment provided proof-of-concept that ethanol production from xylan using a CBP-based approach is indeed possible. cerevisiae strain capable of total hemicellulose hydrolysis. Crous et al. Margolles-Clark et al.

as D-xylulose is a common intermediate in the xylose and arabinose catabolic pathways (Figure 13. 2007b). 1996). Hahn-H€gerdal a a et al. This intrinsic robustness is of crucial importance for implementing industrial-scale lignocellulose conversion to ethanol processes (Almeida et al. 2007a. this was found to be a difficult task. and subsequently be able to reproduce these levels in the recombinant strain rather than seeking to achieve maximum overexpression of all genes (van Zyl et al. most work on pentose-fermenting S. These strains express bacterial arabinose pathways (Figure 13. cerevisiae strains has focused on introducing a functional xylose pathway. 2006.Fuel Ethanol Production From Lignocellulosic Raw Materials Using Recombinant Yeasts 271 activity and product release. Moreover. 2007. 13. 2007). 2007). and distilled alcohol industry. It was therefore initially believed that a xylose-fermenting strain of S. It is perhaps due to this long industrial history and constant strain evolution and selection that S. cerevisiae for achieving efficient fermentation of lignocellulosic raw materials. Since xylose is the preponderant sugar after glucose in lignocellulosic hydrolysates.4) does not result in any appreciable growth nor in ethanol formation (Richard et al. 1980..3 Pentose-Fermenting S. cerevisiae strains have been shown to perform in nondetoxified lignocellulose hydrolysates (Hahn-H€gerdal and Pamment. cerevisiae Strains To date.. cerevisiae is that wild-type strains cannot ferment either xylose or arabinose. cerevisiae could be easily constructed by simply introducing a bacterial xylose isomerase gene. Chiang et al. cerevisiae strains able to efficiently ferment all sugars derived from lignocellulosic raw materials to ethanol (Hahn-H€gerdal et al. 1993). baker’s yeast has an industrial record of several thousand years as an efficient ethanol producer in the brewing. cerevisiae is one of the most stress-tolerant microorganisms. cerevisiae have been generated only very recently (Becker and Boles... cerevisiae expressing xylA (Kuyper et al.4). This was an important observation. On the other hand. In the aftermath of the energy crisis of the 1970s it was recognized that S.. since it had been earlier reported that functional expression of the fungal pathway (Figure 13. Consequently. cerevisiae... 2003). this required extensive adaptation of the engineered strain. Only several years later was the anaerobic ethanolic fermentation of D-xylose reported with a strain of S. A natural limitation of S. 2003. cerevisiae could ferment D-xylulose to ethanol (Wang and Schneider. Wisselink et al. Despite numerous attempts by many different research groups (Table 13. and functional expression of xylA was only achieved with multicopy plasmids . 2004)..4). and continue to be made. arabinose constitutes only a minor fraction in agricultural residues (Hayn et al...2). 2004). only S. and the functional expression of xylose isomerase was not realized until 16 years after the first report on ethanolic D-xylulose fermentation (Walfridsson et al. cerevisiae should in turn be applied to generate efficient arabinose-fermenting strains of S. The strain development and construction strategies discussed in the following paragraphs for xylose-fermenting strains of S. 1981). This is most probably the prime a reason why a large share of research investments have been made. Wiedemann and Boles. 2008). Karhumaa et al. arabinosefermenting strains of S. wine.. It is based on these fundamentals that major research efforts all over the world have been devoted to the construction and development of S. for developing recombinant strains of S. However.

¼ not determined. (1987) Briggs et al. Xanthomonas campestris Bacteroides thetaiotaomicron Piromyces sp.d. n. — 0. in S. (2003) o Grdonyi and a Hahn-H€gerdal (2003) a Kuyper et al.272 Ethanol and Butanol BACTERIA L-arabinose D-xylose L-arabinose Aldose reductase Xylose isomerase ATP NAD(P)H NAD(P)+ FUNGI D-xylose L-arabinose isomerase L-ribulose L-ribulokinase L-arabitol L-arabinitol 4dehydrogenase NAD+ NADH Aldose reductase NAD(P)H NAD(P)+ Lribulose-5-P L-ribulose-5-P 4-epimerase D-xylulose Xylulokinase ATP L-xylulose L-xylulose reductase NAD(P)H NAD(P)+ D-xylulose-5-P Xylitol Xylitol dehydrogenase NAD+ NADH D-xylulose ATP Xylulokinase D-xylulose-5-P Figure 13.d. .0007–0.d. — — — — — — þ þ — þ þ þ þ Sarthy et al. (1989) Amore et al.d.22 — — 0.d. (1984) Amore et al.3–1.d. (1989) Hallborn (1995) Moes et al.14 0.d.d. (2003) Kuyper et al.1 0. Xyl A. cerevisiae Species Activity (U mg protein À1) — n.2 Expression of the xylose isomerase gene.82 0. — n.016 0.012 — n. — — — n. (1996) Walfridsson et al. — Aerobic Additional Reference growth genetic rate modification n. n.005 0.02 n.4 Initial L-arabinose and D-xylose catabolic pathways in bacteria and in fungi Table 13.04 — 0. (1996) L€nn et al. (2005) Ejiofor (2004) Winkler et al. (2007a) Escherichia coli Actinoplanes missouriensis Bacillus subtilis Lactobacillus pentosus Clostridium thermosulfurogenes Thermus thermophilus Thermus thermophilus Streptomyces rubiginosus Piromyces sp. a 0. (2006) Karhumaa et al.

numerous S. Alexander... the industrial performance of recombinant XR-XDH strains is limited by the formation of the byproduct xylitol (Eliasson et al. Xylitol is formed when cells experience a lack of NAD þ (Figure 13. since when xylose is converted to xylitol and secreted into the medium it results in a corresponding reduction in ethanol yield... 2007a). cerevisiae was not sufficient to support D-xylulose catabolism by the recombinant strains (Deng and Ho. The xylA gene in these strains originates from Piromyces sp. Wahlbom and Hahn-H€gerdal. In the meantime. 1986. This is a significant disadvantage. 2002).3-DP-glycerate Glycerol Acetic acid Acetaldehyde Ethanol Figure 13. Enzyme genes that have been overexpressed or deleted (Â) to improve ethanolic xylose fermentation are indicated in capital bold letters . a Overcoming xylitol byproduct formation has been the subject of numerous investigations (Figure 13. a rumen fungus that is an obligate anaerobe (Harhangi et al. cerevisiae strains carry an additional copy of this homologous gene. 1983. 1990). cerevisiae strains expressing the fungal enzymes D-xylose reductase (XR) and xylitol dehydrogenase (XDH) have been constructed (for a recent review. which typically occurs during the limitation or absence of oxygen and other electron acceptors (Bruinenberg et al. 2003).5 Metabolic pathways involved in xylose and glucose catabolism.4).Fuel Ethanol Production From Lignocellulosic Raw Materials Using Recombinant Yeasts 273 (Karhumaa et al. Figure 13..5). 2000. 2007b).. 1988. xylitol formation is reduced and ethanol formation is increased D-xylose GXS1 HXT GFX1 GAL2 Glucose D-xylose XR XI NAD(P)H NAD(P)+ NAD+ XDH NADH ATP GRE3 NADPH NADP+ Glucose Xylitol 6-P-gluconate NADP+ NADPH CO2 G6PDH Glucose-6-P PGI Xylulose XK D-Xylulose-5-P RPE TKL D-Ribulose-5-P RKI Fructose-6-P Ribose-5-P Sedoheptulose-7-P TKL Glutamate Erythrose-4-P NADPH NADP GDH NAD(P)H NAD(P)+ TDH TAL Glyceraldehyde-3-P NAD(P)+ NAD(P)H 2-oxoglutarate 1.4). Therefore almost all xylose-fermenting S. It was recognized at an early stage that the natural a xylulokinase activity of S. Despite this advance.. Notably. see Hahn-H€gerdal et al. Lighthelm et al.

Verho et al.3.. 2002. random mutagenesis-generated industrial strains with superior fermentation performance in nondetoxified lignocellulose hydrolysates have been created (Table 13. 2001.and intermediate-affinity hexose transporters (Hamacher et al. investigations showed that xylitol byproduct formation reflects the intracellular redox balance. 2006). Recently o the facilitated diffusion and proton symporter proteins from the natural xylose-utilizing Candida intermedia PYCC 4715 (Grdonyi et al. Jin and Jeffries. the rate of xylose fermentation is significantly increased.5.. 2006). but that it is also related to the intracellular carbon flux (Hofmeyr and Cornish-Bowden. Furthermore. the rate of xylose utilization by recombinant S. Roca et al. the xylose utilization and fermentation rate is significantly increased (Jeffries et al. Generally. 2007b). is deleted (Tr€ff et al. 2003). 2005). Öhgren et al.274 Ethanol and Butanol when: (i) the ratio of the XR and XDH activities is no greater than 1 : 10 (Eliasson et al... 2001) has frequently been used to improve the performance of laboratory strains that . 1984. 2002).. When individual enzymes of the PPP (Walfridsson et al.... 2006.. (ii) the cofactor specificity of XR is changed to increase its use of NADH (Jeppsson et al.. cerevisiae. 2006.. cerevisiae is much lower than in other yeast species (Gancedo and Lagunas. 1997. Saleh et al. 2007b). 2005) are overexpressed in recombinant xylose-utilizing S. Karhumaa a et al. Wahlbom et al.5). a xylose is transported into the cell by the high.. Fiaux et al. The complexity of generating recombinant pentose-fermenting strains of S. Xylose is metabolized through the pentose phosphate pathway (PPP.. and (vi) the ammoniumassimilating pathway is engineered by exchanging an NADPH-dependent glutamate dehydrogenase for an NADH-dependent isoenzyme (Figure 13. Nevertheless. (v) an NAD(P) þ -dependent glyceraldehyde 3-phosphate dehydrogenase is expressed (Verho et al. 2003. 2003). In the presence of glucose.. 2006). the activity of which in S... which suggests that xylitol formation is not only a function of intracellular cofactor pools. cerevisiae. 2003) or expressing a fusion protein of XR and XDH to improve the cofactor balance of the XR-XDH reaction (Anderlund et al. 1973.. expressing a bacterial transhydrogenase to supply XDH with NAD þ (Nissen et al. evolutionary engineering (Sauer. This approach offers great promise for improving the rate of xylose utilization in S. Jin et al... In addition.. 2002. 2003) were expressed in S. Figure 13.. 2007b).. 2003). 2000). 2003). glucose-mediated enhanced xylose utilization significantly reduces xylitol byproduct formation (Karhumaa et al. which only uses NADPH. 1995) or the entire nonoxidative PPP (Johansson and Hahn-H€gerdal. cerevisiae where xylose transport and metabolism are engineered in parallel.. 2001. Jeppsson et al. 1993).. Meinander and Hahn-H€gerdal. 2003.. cerevisiae strains in defined mineral medium with xylose as sole carbon source is one to two orders of magnitude lower than the rate of glucose utilization (Hahn-H€gerdal et al. a Karhumaa et al. Karhumaa et al. Lee et al. Bro et al. GRE3. 2005. As a direct result. In S.. However. but the affinity of these transporters for xylose is one to two orders of magnitude lower than for glucose (K€tter and Ciriacy. 2001.. cerevisiae has also led the implementation of random strain development strategies to improve growth and ethanol production. 2004. On the other hand.. Karhumaa et al. (iii) the gene for the homologous aldose reductase. 2007b). (iv) the intracellular NADPH pool is reduced by a reducing the activity of the glucose 6-phosphate dehydrogenase gene ZWF1 (Jeppsson et al. cerevisiae a (Leandro et al. 2002. it has also become obvious that xylitol formation cannot be abolished by any single metabolic engineering approach. 2001) have had only a limited influence on changing the pattern of product formation from xylitol to ethanol.

Nonetheless. cerevisiae (Almeida et al. Karhumaa et al.3 also indicate that industrial strains still may require detoxification of the lignocellulose hydrolysate prior to fermentation. there is some evidence to suggest that increasing ethanol tolerance can also provide cross-tolerance to the inhibitors present in lignocellulose hydrolysates (Barber et al. is unlikely to be associated with a single. The ability to confer ethanol tolerance is particularly attractive for microorganisms that have desirable hydrolytic and sugar utilization properties.) and for arabinose (Becker and a Boles. as it now appears. 2006). or at most a discrete number of genes. cerevisiae strains. 2003. the use of auxotrophic or antibiotic resistance markers is not practical for the production of commodity chemicals. cerevisiae have been generated to date (Table 13. Pitk€nen et al.... Furthermore. which suggests that there are still major challenges to overcome in the development of inhibitor-tolerant pentose-fermenting S. research in this area has generated a wealth of information on yeast ethanol tolerance that may eventually lead to the design and construction of strains with improved tolerance to the damaging and inhibitory effects of ethanol. 2005.Fuel Ethanol Production From Lignocellulosic Raw Materials Using Recombinant Yeasts 275 had been metabolically engineered for xylose (Sonderegger and Sauer.. The ultimate goal of generating pentose-fermenting strains of S..3). cerevisiae have been shown to convey inhibitor tolerance to a laboratory strain of S. 2008). 2004. 13.1).4 Lignocellulose Fermentation and Ethanol Inhibition The ability to increase the ethanol tolerance of microorganisms could potentially improve the productivity of lignocellulosic-based processes by decreasing fermentation times... . very little progress has been made on the construction of ethanol-tolerant microorganisms using recombinant techniques. the use of replicating plasmid-carrying strains should be avoided. This observation suggests that inhibitor tolerance can be enhanced not only by natural selection but also by implementing targeted genetic engineering strategies. Therefore. 1992) and are e also found elsewhere (van der Westhuizen and Pretorius. but low ethanol tolerance. In spite of this. 1992). The data in Table 13. in order for strains to be useful in industrial applications genetic stability is a critical attribute. Dehydrogenases isolated from an inhibitor-tolerant industrial strain of S. consequently. with potentially considerable gains in ethanol yields being realized when the solids loadings in hemicellulose hydrolysate-fed fermenters are increased..2). which reflects the point that targeted genetic manipulation is much more complicated for diploid and aneuploid strains. Furthermore. Relatively few recombinant industrial pentose-fermenting strains of S. Likewise. cerevisiae is to implement such strains in large-scale industrial fermentation of lignocellulose hydrolysate (Figure 13. 2005. Such strains can be isolated from industry (Lindn et al. Kuyper et al. 2002). Wisselink et al. One approach to developing more ethanol stress tolerant yeast strains is to improve the protective response elicited by cells during ethanol stress. pentosefermenting pathways must be introduced in industrial strains that inherently are tolerant to the inhibitors formed during pretreatment and hydrolysis of the lignocellulosic raw material (Figure 13. Laboratory strains are generally too sensitive to the growth inhibitors that these hydrolysates typically contain (Karhumaa et al. 2007) fermentation. 2003. the reason being the complex nature of ethanol tolerance which.

(2005) a TMB3400 TMB3400 424A LNH-ST 424A LNH-ST MT8-1/Xyl/BGL Adapted TMB3006 Spruce TMB3006 Spruce TMB3400 Hahn-H€gerdal and a Pamment (2004) Hahn-H€gerdal and a Pamment (2004) a Recalculated from reference. (2006) Sedlak and Ho (2004) Sedlak and Ho (2004) Katahira et al.24 — — — — 0.41 0.276 Table 13.66 0. (2006) ¨ Ohgren et al.1 Fed-batch F12 ??? Batch Olsson et al.30 0.41 0. (2006) ¨ Ohgren et al.27a 0.42 — 0.44 0.3 Detoxification step Cultivation arrangement Specific ethanol productivity (g lÀ1 hÀ1) 0. D ¼ 0.32 0.37 0. .41 0.005–0.25 0. (2006) Hahn-H€gerdal et al. cerevisiae strains Reference Ethanol and Butanol Hydrolysate source Strain Still bottoms fermentation residue (vinasse) Corn stover Corn stover Corn stover Corn stover Wood chips Northern spruce 70 % — — Overliming ??? Overliming — — — Fed-batch Batch SSF Fed-batch SSF Batch Batch Batch Continuous.43 Ethanol yield (g g total sugarÀ1) Fermentation of lignocellulose hydrolysate with industrial xylose-fermenting S.

1995b). Although limited. (iii) reductions in growth. Hounsa et al.. of these gene products. Mishra. 1987. cerevisiae Exposure to ethanol stress causes many changes to occur in yeast.. 1998.. leading to increased fatty acid length and increased proportions of unsaturated fatty acids and sterols in yeast cell membranes (Beaven et al. 1999). Similarly to what is observed in many microorganisms. 1989. see D’Amore et al. Sales et al. Betz et al.. 1993.... have led to doubts concerning the role of trehalose in protecting cells from stress (Nwaka and Holzer. 2004. The localization of Rat8p does not change in heat-shocked cells. but was inducible in the wild-type. (iv) nutrient intake and ATPase activity (Ingram and Buttke. which suggests the . cerevisiae hsp12D knockout mutant as compared to the wild-type when these strains were grown in 10–12 % ethanol. HSP70 (Piper et al. 1998). However. HSP26. 1992) and Hsp12 (Sales et al. HSP82. Soto et al.. 1995). A number of signaling pathways appear to be activated in the S. Parsell et al. Yeast also accumulate intracellular trehalose. Sanchez et al. including: (i) an increased frequency of petite mutations. (2000) observed a reduced growth rate in an S.. 1984. Walker. and other findings. 1996. (ii) the inhibition of metabolism. 1994).. Likewise. (1992). yeast have evolved protective adaptive responses to the effects caused by ethanol exposure.4. Varela et al. (2004) observed that ethanol stress as well as heat shock causes selective mRNA export. 2000) have been shown to influence yeast tolerance to ethanol. 1991. when using a hsp104D yeast mutant. This. 1995a. and HSP12 (Praekelt and Meacock. HSP30 (Piper et al.. An increased expression of HSP genes has been observed when yeast is exposed to ethanol stress. demonstrated that heat-induced tolerance to 20 % ethanol could not be achieved in the mutant.Fuel Ethanol Production From Lignocellulosic Raw Materials Using Recombinant Yeasts 277 13. moreover. 1994. cerevisiae response to ethanol challenge. 1982). Bulk poly(A)þ mRNA accumulates in the nucleus. However. Takemura et al. 1990. only Hsp104 (Glover and Lindquist. 1995). leading to suggestions that trehalose may function as a cellular protectant towards stress. 1990. It was found that the nuclear localization of DEAD box protein Rat8p changed rapidly and reversibly in response to ethanol stress.. Kim et al. Jones. cerevisiae to environmental stress. 1998. Nwaka et al. Recent studies in this area have identified novel ethanol-specific responses (Takemura et al. Sanchez et al. 1994). One of the earliest recognized ethanol stress adaptive responses is an alteration in the fatty acid profile of cellular membranes. this may have interfered with other cellular functions that are important for stress tolerance... as argued by Singer and Lindquist (1998). Piper. it has been observed that yeast mutants lacking the NTH1 gene (this mutation causes the accumulation of intracellular trehalose) are compromised in their ability to survive extreme heat (Nwaka et al.. it should be noted that the Nth1D mutant accumulated excessive trehalose levels and. and (v) disruption of the membrane structure leading to increased membrane permeability and subsequent loss of electrochemical gradients and membrane-associated transport activities (for reviews. In particular. Contrary to its protective role. the expression of genes coding for heat shock proteins (HSPs) is a well-documented response of S. 1992).. in both cases the influence of these gene products on ethanol tolerance was only slight. Sanchez et al. the more widely recognized genes being HSP104 (Piper et al. 1988). whereas mRNA of HSPs is exported under such conditions. potentially by stabilizing proteins and preserving the integrity of cellular membranes (Attfield. 2004)..1 The Ethanol Stress Response of S. This change correlated strongly with the blocking of bulk poly(A)þ mRNA export caused by ethanol stress.

1996). The localization of Asr1p in the nucleus is exclusive to alcohol stress. 2006). these two ethanol-specific responses raise the possibility that S. possibly reflecting growth arrest during stress to facilitate energy conservation and cellular adaptation (Chandler et al. (2004) to propose that yeast subjected to ethanol stress enter a pseudo-starvation state.2 S. with all reports observing the upregulation of genes associated with the trehalose pathway (TPS1. and in all of these only small changes in the ethanol tolerance of the mutant strain were observed.. cerevisiae possesses a signal transduction pathway that is specific for ethanol. SSE2). SSA2. HOR7 and DAK1) were also found to be upregulated during ethanol stress (Alexandre et al. It should be emphasized here that this pattern is not observed during any other stress conditions tested to date. HSP78. with gene array-based studies in yeast undergoing ethanol stress reporting the upregulation of glycolysis-associated genes (GLK1. Chandler et al... or the cells lack sufficient glycolytic activity.. ALD4 and PGM2). cerevisiae strain that overexpressed both the D9-fatty acid desaturase gene (OLE1) from . SSA3. 2004. but this is yet to be tested. This protein has been shown to constitutively shuttle between the nucleus and cytoplasm under normal conditions. SSE1.278 Ethanol and Butanol presence of an ethanol-specific response in yeast. Fujita et al.. and high-affinity hexose transporter genes (HXT6 and HXT7) (Alexandre et al. even though ample glucose may be present in the medium. such as oxidative. As a result. 2004. 2004. HSP26. Another result from array studies is the downregulation of genes associated with protein biosynthesis.. 2004). These results led Chandler et al. It was proposed that Asr1p is involved in a complex signal transduction pathway that enables yeast to acclimatize to ethanol stress. and to accumulate in the nucleus upon exposure to alcohol (Betz et al. and TLS1). 13. TPS2. cerevisiae confirmed earlier findings on the ethanol stress response.. Chandler et al. the transport of specific mRNAs out of the nucleus is modulated during ethanol stress in yeast (Saavedra et al. It was proposed that the nuclear localization of Rat8p contributes to the selective export of mRNA in ethanol-stressed cells.. HXK1. 2004a). Izawa et al. and that this particular response in the export of mRNA is unique to ethanol stress (Takemura et al. 2001. Gasch et al. and HSP104 being among the most highly upregulated (Alexandre et al... No unique phenotype has yet been identified for asr1D strains during ethanol stress (Betz et al. Notably. Gene array studies on S. It is important to note that many genes with unknown function have altered expression levels during ethanol stress. (2000) constructed an S. These studies also report the ethanol-induced upregulation of members of the HSP70 family (SSA1. as described earlier. 2004). 2006). Caution must be taken in interpreting these results however since. GRE3. TDH1. with HSP12. cerevisiae Mutants with Altered Ethanol Tolerance Very few studies were performed that made use of overexpression systems to enhance ethanol tolerance in yeast. One protein that has recently been suggested to have a signaling role specific to the ethanol stress response is Asr1p (Alcohol Sensitive Ring/PHD). 2006.. 2001). cell growth and RNA metabolism. Kajiwara et al.. 2001. SSA4. and genes encoding HSPs. HOR2.4. It is becoming clear that cellular energetics are particularly affected by ethanol exposure.. Other genes associated with energy recovery and conservation (GPD1. 2004). osmotic.. 2000). nutrient limitation or heat stress (Betz et al. Izawa et al. 2004.. van Voorst et al.. the impact of ethanol on cell function either impedes glucose transport.

.. SMI1. this recombinant S. (2004) Fujita et al. while Kubota et al. cerevisiae to identify genes required for ethanol tolerance (van Voorst et al. v/v) and Kubota et al. The results of these studies show very little commonality with regard to the effects of individual genes on ethanol tolerance. Genes that impact on the cytoskeleton. 2006). although the different conditions and selection criteria used is likely to explain many of the discrepancies. 2001). Kubota et al. (2004) (11 %. In another similar study.. v/v) to screen the ethanol tolerance of S.Fuel Ethanol Production From Lignocellulosic Raw Materials Using Recombinant Yeasts 279 S. these groups identified 137 and 256 open reading frames (ORFs) that provide tolerance to these ethanol concentrations. Ethanol has been shown to disrupt the yeast cytoskeleton.. Mitochondria are most likely required for the removal of reactive oxygen species (ROS) generated from ethanol exposure (Costa et al. 1993). Fujita et al. In the presence of 15 % (v/v) ethanol. The number of genes identified uniquely or commonly is shown. Many genes required for vacuolar and mitochondrial function were found to be associated with ethanol tolerance. VPS36.6 Comparison of the results from three independent deletion library screens of S. Respectively. GIM4) being commonly identified in all three studies (Figure 13. Interestingly. only 10 % of the cells in the wild-type strain culture were found to be viable. this strain is not only able to synthesize dienoic fatty acids but it also has higher unsaturated fatty acid content as compared to the parent strain. cerevisiae knockout strains.6). (2006) (10 %.. whereas vacuolar function is anticipated to be necessary for pH homeostasis (Fujita et al. (2006) 178 56 4 17 7 70 18 van Voorst et al. Several genome-wide screens using yeast single knockout strains have been performed with S. High ethanol concentrations were used by Fujita et al. (2006) isolated 46 mutants sensitive to 6 % (v/v) ethanol. cerevisiae strain exhibits a higher survival rate than the wild-type strain. 2004). Takahashi et al. (2006) Figure 13. 2004). These results show very little commonality across the three studies with regard to the effects of individual genes on ethanol tolerance . the results from these studies suggested a strong correlation between cell membrane genes or cell wall architecture genes and ethanol tolerance (van Voorst et al. 2006. 2006. 2006. with only four genes (GIM5. Based on a total lipid analysis.. As predicted based on previous physiological and genetic studies. with 40 % of the population remaining viable after 6 h exposure. cerevisiae and the D12-fatty acid desaturase gene (FAD2) from the plant Arabidopsis thaliana. morphogenesis and the cell cycle were also identified as being required for ethanol tolerance in these genome-wide screens. In comparison.. van Voorst et al. which in turn has been proposed to activate the morphogenesis checkpoint and delay the cell cycle (Kubota et al. cerevisiae genes associated with ethanol tolerance..

an ethanol challenge provided the basis of the selection pressure that was applied in both cases (Stanley et al. a faster and more complete glucose utilization.280 Ethanol and Butanol the functional categories of the ethanol tolerance genes often support the global gene expression studies. while the other mutant was generated using an evolutionary engineering approach. In this latter method.. These results strongly support the view that the vacuole plays a major role in ethanol tolerance. This can be particularly well exemplified by a study where two ethanol-tolerant mutants of S. Cakar et al. also identified in the genetic screens and expression studies. however. and an increased ethanol yield under a . There are surprisingly only a few reports on the creation and isolation of ethanol-tolerant mutants. 2005). 1988). cerevisiae Strains Most research to date on improving ethanol tolerance in yeast strains has been conducted in the belief that such improvement will lead to higher ethanol productivities and yields (Jimenez and Benitez. 2007). VPS16 and VPS28. The identification of QTL in the region of HXK1 and PFK26 reinforce the interpretation that ethanol causes a pseudo-starvation state.. cerevisiae were created. Alper et al. One mutant was generated using EMS mutagenesis. 2004). This may once again be related to the earlier-described observation that the transport of specific mRNAs out of the nucleus is modulated during ethanol stress. 13. The best-performing isolate displayed a prolonged exponential growth phase. the binding preferences of key global transcription factors are modified by a combination of mutagenesis and selection. followed by selection for ethanol-tolerant phenotypes using serial subculturing in 6 % (v/v) ethanol.4. Both isolates were significantly more ethanol-tolerant according to their growth profiles. followed by chemostat-based selection. An increase in ethanol tolerance of isolates according to their growth profile or CO2 output could thus be observed. An incomplete knowledge of the key cellular mechanisms underpinning ethanol tolerance means that there are few successful targeted approaches to generating ethanol-tolerant variants. Two vacuolar protein sorting genes. locate within two of the five identified QTL. One successful such nontargeted approach is adaptive evolution in continuous cultures using ethanol as the selection pressure. since selecting mutants on the basis of their ethanol tolerance may not necessarily lead to increased ethanol productivity or yield. (2006) used ‘global transcription machinery engineering’ to isolate ethanol tolerant strains. cerevisiae genome that can be correlated to the difference in ethanol sensitivity between two strains (Hu et al. the majority of the genes that are specifically induced by ethanol do not confer increased ethanol sensitivity when deleted. but a more thorough analysis of the phenotypes was not conducted (Brown and Oliver.. suggesting that the connection between gene expression and phenotype during ethanol stress may not be straightforward.. A more recent study used mapping of quantitative trait loci (QTL) to identify regions in the S. this is otherwise known as ‘evolutionary engineering’ (Sauer. their ethanol productivity and yields were similar to those of the parent strain. 2004). 1982. Mutations in the TATA-binding protein gene SPT15 are introduced using the polymerase chain reaction (PCR). 2001).3 Approaches Used to Develop Ethanol-Tolerant S. This is important to note as the relationship between selection conditions and the phenotype of the isolates can often be counter-intuitive (Stanley et al.

cerevisiae strains that perform efficiently in large-scale industrial fermentations. and the challenges faced in attempting to increase ethanol tolerance in strains using recombinant DNA techniques. The desired phenotype was shown to be due to three mutations in the SPT15 gene that appeared to alter the gene product’s interaction with Spt3p.iogen. however. xylose and arabinose. . This observation suggests that each gene encodes a necessary component of a complex. Indeed. interconnected network that supports the ethanol-tolerant phenotype.5 Perspective Considerable progress has been made towards our understanding of genetic and metabolic functions in S. the identification of an optimal combination of saccharolytic enzymes. a subunit of the SAGA histone acetyltransferase that regulates a number of RNA polymerase II-dependent genes. There is no doubt that economic gains can be achieved in the conversion of lignocellulose to ethanol by using a CBP approach. Whereas. Three factors appear to be particularly critical to succeed in this endeavor: (i) the ability to increase the expression of cellulases in recombinant yeasts. ethanol tolerance in S. and how these may be modified to improve the performance of lignocellulose-based fermentations. (ii) . cerevisiae. This promise will continue to stimulate research on the development of CBP-capable S. The priority. this is where the research focus has been over the past 20–30 years. In fact. and developing ways of improving. and (iii) the engineering of catabolic pathways that enable the concomitant utilization of glucose. cerevisiae strains. increasing our understanding of. and on the other hand the availability of global transcription machinery engineering techniques to create yeast strains with improved ethanol tolerance and productivity. given on the one hand recent findings regarding the possible existence in yeasts of signaling proteins that are ethanol stress-specific. The creation and isolation of ethanol-tolerant mutants is evidence that genetic engineering approaches can potentially be used to improve yeast ethanol tolerance. These studies demonstrate the complex nature of ethanol tolerance in microorganisms. this knowledge may not be far away. Similarly. and will continue to be for some time. While the overexpression of these genes individually did not result in the desired effect. Knowledge of the molecular events that occur in yeast during ethanol stress has improved substantially in recent times. cerevisiae and other organisms will potentially lead to higher fermentation productivities and ethanol yields. recombinant xylose-fermenting strains of S. the development and application of S. cerevisiae are currently being used in pilot-plant operation (www.Fuel Ethanol Production From Lignocellulosic Raw Materials Using Recombinant Yeasts 281 number of different conditions and glucose Microarray analysis of the spt15 mutant demonstrated the overexpression of a number of Spt3p-dependent genes with broad function. cerevisiae strains with improved ethanol tolerance and CBP ability still lies in the future. 13. many of the most highly overexpressed genes were essential for the spt15dependent tolerance. in the development of recombinant yeasts for lignocellulose hydrolysate fermentations is in the generation of pentosefermenting S. The design of ethanol-tolerant yeast strains will however remain an elusive goal until a better understanding is acquired of the key molecular mechanisms underpinning ethanol stress tolerance.

395–406 (1995).. Appl. Environ. P.H.C. Oliver. J. 25.R. Production and tolerance of ethanol in relation to phospholipid fatty-acyl composition in Saccharomyces cerevisiae NCYC 431.. Microbiol. 82. van Weelden. 8.W. Gen. Alexander. J. A. Bruinenberg. Modig. Hackstein. P. 1447–1455 (1982). Molecular cloning. J.J.282 Ethanol and Butanol 13. 98–103 (2001). Boles.. Briggs. 25. o e NADPH-coupled reduction of 5-hydroxymethyl furfural (HMF) and its implications on product distribution in Saccharomyces cerevisiae.H. Boxma.J. van Dijken and W.-F. 3. E. Hollenberg. a Increased tolerance and conversion of inhibitors in lignocellulosic hydrolysates by Saccharomyces cerevisiae. Biotechnol. B. G. Chem.. N. C. Ricard. 939–945 (2008). Hahn-H€gerdal. 4144–4150 (2003). R. Voncken. J.. Beaven. Blondin. Brown and S. thanks the Swedish National Energy Administration for financial support.vs. Fink and G. S. Technol. Metab. K. A. a novel yeast ring/PHD finger protein. Rose. Microbiol. Eur. 25. In silico aided metabolic engineering of Saccharomyces cerevisiae for improved bioethanol production. Microbiol.J.R. Almeida. Tielens and J. G. Charpentier and A. Dien. Acetone stimulation of ethanol production from D-xylose by Pachysolen tannophilus. Akhmanova. Adam. Biotechnol. N. H. Forster and J. Biotechnol.A. 867–875 (1999). 102–111 (2006). Induced expression of bacterial b-glucosidase activity in Saccharomyces. DNA structure and expression of the Escherichia coli D-xylose isomerase. Nichols and B. Mol.G. M. 11. Microbiol. T. A.S. The role of redox balances in the anaerobic fermentation of xylose by yeasts. T. 69. B. Microbiol. FEBS Lett. Rubio-Texeira and J. Almeida. Appl. Petersson. R. Barber. Prog. Gorwa-Grauslund. Bro. Hartley. Schlenstedt and S. A. Nucleic Acids Res. Attfield. W. Engineering yeast transcription machinery for improved ethanol tolerance and production. . 498. Eng.. H. Metab. Scheffers. Alexandre. Expression of bifunctional enzymes with xylose reductase and xylitol dehydrogenase activity in Saccharomyces cerevisiae alters product formation during xylose fermentation. Ansanay-Galeote.N. S. Becker and E. Lidn and M. Appl. Biol. J. Science. J.E. Global gene expression during shortterm ethanol stress in Saccharomyces cerevisiae. J.J.R. 3. Fermentations with new recombinant organisms. Microbiol. 104–108 (2000).... Trehalose accumulates in Saccharomyces cerevisiae during exposure to agents that induce heat shock response.R. 119–122 (1982). S. Gorwa-Grauslund.S. J. A. 314. Anderlund. Polaina. Appl. References A. Modig. Nielsen. Nevoigt. 78. V. Acetaldehyde stimulation of the growth of Saccharomyces cerevisiae in the presence of inhibitors found in lignocellulose-to-ethanol fermentations.. C.. B. J. B.B. Bailer. Microbiol. 1389–1399 (2004). Peter.. 611–616 (1984). 18. G. Asr1p. Biotechnol. 225. Yeast. 259–263 (1987).G. Biotechnol.M. Huynen.. M. Liden and M. Pamment.M. Ind. J.P. signals alcohol stress to the nucleus. 340–349 (2007). 279. H. Chem. Dequin and B. C.6 Acknowledgments The authors thank Christer Larsson for excellent editorial assistance.. Bothast. Hansson and N. J.H. T.V. Stephanopoulos. H.M. 7515 (1989).A. van Hellemond. NADH. Alper. 28174–28181 (2004).. Amore and C.. Hahn-H€gerdal. Laadan. S. Biotechnol. The anaerobic chytridiomycete fungus Piromyces sp. 203–207 (1986). F. G.H. 15. Eng. Betz.P. R€der. B. Jannink. Biotechnol. M. Isolation of ethanol-tolerant mutants of yeast by continuous selection. Microbiol. Xylose isomerase from Actinoplanes missouriensis: primary structure of the gene and the protein. Appl. 287–292 (1983). 16. P. E2 produces ethanol via pyruvate:formate lyase and an alcohol dehydrogenase E. 1565–1568 (2006).W. A modified Saccharomyces cerevisiae strain that consumes L-arabinose and produces ethanol. 226–235 (2001). Radstr€m and B.-F.  o a M. 128. J. FEBS Lett. 51. EMBO J. Moxley. van Alen. Regenberg.. Lancashire and B. G.

A. Microbiol. 467–473 (1995). M. Microbiol. Sonderegger. Dien.S. Tsao. 23–30 (1999). A study of ethanol tolerance in yeast. V. Biotechnol.T. Y. Environ.H.C. Stewart. Biochem. Cloning and expression of an Aspergillus kawachii endo1. Appl. Functional expression of cellobiohydrolases in Saccharomyces cerevisiae towards one-step conversion of cellulose to ethanol. I. Rev. Anaerobic xylose fermentation a by recombinant Saccharomyces cerevisiae carrying XYL1. 42. Enzyme Microb. Comprehensive gene expression analysis of the response to straight-chain alcohols in Saccharomyces cerevisiae using cDNA microarray. Eddy and S. Pretorius and W. A. Technol. Expression and secretion of Clostridium thermocellum endoglucanase A gene (celA) in different Saccharomyces cerevisiae strains. The holy grail of consolidated bioprocessing. 569–578 (2005).S.S. den Haan. Technol. J. Deanda. C. 288–297 (2001). Costa. 258–266 (2003). Eukaryot. L.G.H.. Sonderegger and U. Crous. 25. Microbiol.M. 57–67 (2004a).E. Microbiol. van Zyl. Tamerler. Crit. M. Cotta and T. Biotechnol. J. Chen and G.A. Eng. X. J. I. Eliasson.. Enzyme Microb. Fiaux. C. Technol. 300.Fuel Ethanol Production From Lignocellulosic Raw Materials Using Recombinant Yeasts 283 Z. Chung. Bacteria engineered for fuel ethanol production: current status. A genomic approach to defining the ethanol stress response in the yeast Saccharomyces cerevisiae.C. 284–289 (1981). M. Rose. Reis. Hofmeyr. A.W.H. P. Fujita. G. Seker.W.J.M. Characterization of the Aureobasidium pullulans aglucuronidase expressed in Saccharomyces cerevisiae. Enzyme Microb.S.4-b-xylanase gene in Saccharomyces cerevisiae. Ho. Matsuyama. Appl. Appl. Biotechnol. Y. Kang. I. Cho. 63. Pedler and B. 24–25. FEMS Yeast Res.. Biochem. Lynd and W. 503–506 (1997). den Haan.Sc. D. Hydrolysis and fermentation of amorphous cellulose by recombinant Saccharomyces cerevisiae. 66. La Grange. de Wet.H. Kim. Jeffries. L..S. Han and S.K. J.-H.W. Quintanilha and P.F. Cakar. Cell.S.. A. Deng and N. Nam. Curr. Russell and G.. W. thesis. K. Sauer. Biotechnol. van Zyl. M... S.. 3381–3386 (2000). K. Ethanol Producer Magazine. XYL2. R. C. Technol. Evolutionary engineering of multiple-stress resistant Saccharomyces cerevisiae.H. Prior. Microbiol. van Zyl.M. Chiang. Eliasson.. Gong.K. Lett. D.. J. D.G.A.X.M. C. Crous. K.. Microbiol. 9..S. November (2007). B. U. Appl.J. Cakar. Biotechnol. Wuthrich. Environ..D. Ann. d-Integration of endo/exo-glucanase and b-glucosidase genes into the yeast chromosomes for direct conversion of cellulose to ethanol. Moradas-Ferreira.. 4465–4470 (1996).. Development of an arabinose-fermenting Zymomonas mobilis strain by metabolic pathway engineering. 29. and XKS1 in mineral medium chemostat cultures. 608–614 (1993).J. Biophys. B. 62. E. Chambers.. Szyperski and U. McBride. J. van Zyl. 5. D-xylulose fermentation to ethanol by Saccharomyces cerevisiae. Yoo and H. 170–180 (2003). Metabolic-flux profiling of the yeasts Saccharomyces cerevisiae and Pichia stipitis. Arch. D’Amore. Molecular tools for improving xylose fermentation in xylose isomerase expressing yeasts. T. 287–304 (1990). 46. 256–260 (1996). L. Pretorius and W. 2. 40. 75–102 (2004). Z. 97. Wahlbom and B. B. Nam. C. Rogers and P. Metab.J. Genet.W. Sauer.H.R. Appl. Iwahashi. R. Lund University (2004). Ebert. 193–199 (1990).P.P. T. 28. Picataggio. C. van Zyl and B. Chandler. Lynd and W. Microbiol.F. 19. 649–656 (2006). Stanley. K. Appl. J.R. M. Appl. L. 54. M. Zhang.O. Christensson. 38. Enzyme Microb. Kobayashi and H. . The xylose reductase/xylitol a dehydrogenase/xylulokinase ratio affects product formation in recombinant xylose-utilising Saccharomyces cerevisiae. Environ. Hahn-H€gerdal. Cloning and expression of the a-L-arabinofuranosidase gene (ABF2) of Aspergillus niger in Saccharomyces cerevisiae. 87–94 (2007b). S. 1291–1299 (2007a). Acquisition of ethanol tolerance in Saccharomyces cerevisiae: The key role of the mitochondrial superoxide dismutase. Shin. Panchal.A. Hahn-H€gerdal. Ejiofor.H. I. J. 9. C.. Xylulokinase activity in various yeasts including Saccharomyces cerevisiae containing the cloned xylulokinase gene.

2783–2788 (2002) H. 41–65 (2007). Rodrigues.. 471–475 (1984). Morikawa. Hahn-H€gerdal. 108. Construction of whole-cell biocatalyst for xylan degradation through cell-surface xylanase display in Saccharomyces cerevisiae. Fukuda and A. Hahn-H€gerdal.. A. Appl. Biotechnol. E. High capacity a a xylose transport in Candida intermedia PYCC 4715.P. Appl. 6. Y. Biotechnol.-F. 11. Hahn-H€gerdal and N.H. Genet.B. Ph. Fujita. 1207–1209 (2004). Hamacher. C. Katahira. in Bioconversion of forest and agricultural plant residues. Okada. C. B. Y. 73–82 (1998).3-1. Karhumaa. Microbiol. Mol. 94.U. Adv. Microbial pentose metabolism. Cell. Hahn-H€gerdal. 32. The Streptomyces rubiginosus xylose isomerase is misfolded a a when expressed in Saccharomyces cerevisiae..-F. Y. Sinner and H. Synergistic saccharification. FEMS Yeast Res. Fact. Gasch.-F. 134–141 (2003). Pamment. 180.B. 45–52 (2003). K. Esterbauer. Grdonyi. 189–195 (2002a). Glover and S. 8. Hahn-H€gerdal. Saddler (ed. 3. Eisen. Biotechnol. Hespell. Wyckoff. W. 68. Ueda.D. Microbiol. 4594–4597 (1996). CAB International. Metabolic engineering a for pentose utilization in Saccharomyces cerevisiae. Galbe and G. 1207–1212 (2004b). FEMS Yeast Res.T. J. O. Bothast. Hsp70. Appl. I. Klinger. Karhumaa. a 113– al.P. Ueda. M. 147–177 (2007b). Emmens. thesis. K.J. C. Grdonyi. Kondo.284 Ethanol and Butanol K.M. J. Catal..). 4241–4257 (2000). M.S. J. Pretreatment of lignocellulosic materials for efficient bioethanol production. H. C. H. Role of cultivation media in the development of yeast strains for large scale industrial use. Kawaguchi. M. Tanaka. I. M. Wallingford UK (1993).. a Towards industrial pentose-fermenting yeast strains. M. Becker. Hallborn. 62. Gancedo and R. 148. de Laat. Takahashi. Gorwa-Grauslund. Biochem. Fukuda and A. J. Harhangi. M. J. Cell. Pronk and H. Microbiol. van a o Zyl. Hinchliffe and W. M. Ito. Y. 74. M. 193–200 (1973). 5136–5141 (2002b). Dien and R. P. Mol. H. Boles. Hsp104. Microb. Metabolic engineering of Saccharomyces cerevisiae: Expression of genes involved in pentose metabolism. Hahn-H€gerdal. Y. Zacchi. H. B. M..R.S. Kao. Plant Sci. of amorphous cellulose by use of an engineered yeast strain codisplaying three types of cellulolytic enzyme. R. Gorwa-Grauslund. A. Tanaka. Ueda. M. B. Eng. Cell. Y. R. Grdonyi and B. H.R. Arai. Gorwa-Grauslund. Biochem. Direct and efficient production of ethanol from cellulosic material with a yeast strain displaying cellulolytic enzymes. and Hsp40: A novel chaperone system that rescues previously aggregated proteins. Kondo. Hayn. Jeppsson and M. B.G. Kobayashi and H. Fujita. Characterization of the xylosea a transporting properties of yeast hexose transporters and their influence on xylose utilization. 70. Hahn-H€gerdal and E. Steinm€ller. M. 4.. Contribution of the pentose phosphate pathway to glucose metabolism in Saccharomyces cerevisiae: a critical analysis on the use of labelled glucose. Lund University (1995). R. Steiner.4-b-glucanase gene of Bacillus subtilis in Saccharomyces cerevisiae. J. S. Spencer-Martins and B. Okada. Biochem. B. Box. Basic research and pilot u plant studies on the enzymatic conversion of lignocellulosics. Enzyme Microb.. 108. A. 744–750 (2006). Appl. Microbiol. Technol. Lett. J. W. A. T. Expression of the cloned endo-1.. van der Drift. Arch. Fukuda and A.M. T. Larsson.... 17.T. Appl. and direct fermentation to ethanol. C. 252–259 (2003).. Environ. Stabilization of pet operon plasmids and ethanol production in Escherichia coli strains lacking lactate dehydrogenase and pyruvate formatelyase activities. strain E2 follows the bacterial pathway. S. Adv. 31 (2005). B. 937–953 (2007a). Iwahashi.. Kondo. G€rgens and W. K. J. The genome-wide screening of yeast deletion mutants to identify the genes required for tolerance to ethanol and other alcohols. H..T. Genomic expression programs in the response of yeast cells to environmental changes.N. Morikawa. Environ. J. Jetten. ..B. Biotechnol. Carmel-Harel. Karhumaa. Akhmanova.S. M. A. H. Spellman. Matsuyama. Xylose metabolism in the anaerobic fungus Piromyces sp.J. Spencer-Martins and M. Biol. J. M. Fonseca. Microbiology. Environ. 1. Osterberg. M. Eng. Microbiol. M. Fujita. Curr. Op den Camp. Lagunas. Fujita. van Dijken. Lindquist.

H. Cornish-Bowden. T. Ingram. Selection of ethanol-tolerant yeast hybrids in pH-regulated continuous culture. H.R. 17. S. Engineering yeasts for xylose metabolism. Microbiol.N. T. 1839–1843 (2000) K.V. 25. 27. Saccharomyces cerevisiae engineered for xylose metabolism exhibits a respiratory response. Hahn-H€gerdal and M... 665–673 (2006). Bioeng. Appl. Izawa. Environ. an alcohol-responsive factor of Saccharomyces cerevisiae. 175. Jeffries. 47–51 (2000). Biotechnol.. Jimenez and T. B.M. 560–565 (2006).G.P. E.P. is dispensable for alcoholic fermentation.H. Microbiol.M. Environ.. M. Appl. Tamaki.W. Technol. M. Biotechnol. T. B.O. Physiol. Investigation of limiting metabolic a steps in the utilization of xylose by recombinant Saccharomyces cerevisiae using metabolic engineering. 434–441 (2001). 277–286 (2003). Lee. Kumagai. D. 2420–2425 (1987). Eng. 54. T. Akiba. H. Microbiol. Sewell and J. 93. C. Utilization of xylose by bacteria. 320–326 (2006). S. Environ.. 476. Jeffries. 53.W. Microbiol. The level a of glucose-6-phosphate dehydrogenase activity strongly influences xylose fermentation and inhibitor sensitivity in recombinant Saccharomyces cerevisiae strains. 92. Zhang and Z. Biotechnol. J. Karhumaa. 253–300 (1984). 171–176 (1984). Kita and Y. 1479–1487 (2007).. S.4-glucanase from Thermoascus aurantiacus and its expression in yeast. Wang.. Jin.. 1604–1609 (2002). Microb. Yeast. J. R. H.. Improved ethanol tolerance of Saccharomyces cerevisiae strains by increases in fatty acid unsaturation via metabolic engineering. Leach. J. The a expression of a Pichia stipitis xylose reductase mutant with higher K(M) for NADPH increases ethanol production from xylose in recombinant Saccharomyces cerevisiae.Grauslund. Hahn-H€gerdal. Yeast. Hofmeyr and A. 657–661 (2003). J. Kajiwara. . 1263–1272 (2003). 70. Microbiology. Yamamoto and H. Ingram and T. Tan. Y. Benitez. 22.M. Hahn-H€gerdal and M.4-glucanase from Aspergillus niger and its expression in yeast.. 105–108. Ikeda.. Luo. Tamaki. Jeppsson.W. FEBS Lett. Bioeng. Hong. Cloning of a gene encoding a highly stable endo-b-1. Genetic dissection of ethanol tolerance in budding yeast S. Jeppsson. Appl. Yang. J. Suga. Hahn-H€gerdal and M. M. P. and fungi.A. 25. Bioeng. 22. Gorwa.. B. Prior..W. Hohmann and B. L. Hong. Adv.W. Hu. Role of trehalose in survival of Saccharomyces cerevisiae under osmotic stress. Curr. Yamamoto and H. Enzyme Microb. Biosci. Opin.. Jin and T. Lightfoot.-F. FEMS Yeast Res. K. Jensen. J. B.-F. Gorwa-Grauslund. Sone and K. 72. Changing flux of xylose metabolites by altering expression of xylose reductase and xylitol dehydrogenase in recombinant Saccharomyces cerevisiae. Franke. Fady and E. 130–153 (1989). Adv. Laplaza and T. Lett. L. X. R. T. Jones.S. Jeffries.W. K. Jeffries. K. Hahn-H€gerdal and M. Appl. Cloning of a gene encoding a thermo-stable endob-1.-F.. 68. 6816–6825 (2004). 144. Appl. 11. 27. Biochem. Effects of alcohols on micro-organisms. The non-oxidative pentose phosphate pathway controls the a fermentation rate of xylulose but not of xylose in Saccharomyces cerevisiae TMB3001. Hounsa. Asr1. L. Biotechnol. Inoue. Preston. M.O.W. Kumagai. J. 917–922 (1988). T. Jeppsson. Buttke.S. 277–282 (2002).-F. Genetics. Lett. B. Thevelein. 671–680 (1998). Biotechnol. G. Johansson and B. 359–368 (2005). Biochem.F. Gorwa-Grauslund. 1–32 (1983). Conway. B. Biotechnol. Reduced oxidative a pentose phosphate pathway flux in recombinant xylose-utilizing Saccharomyces cerevisiae strains improves the ethanol yield from xylose. Johansson. K. Regulating the cellular economy of supply and demand. Biotechnol.. Gorwa-Grauslund. Johansson. Effect of glucose supplements of the fermentation of xylose by Pachysolen tannophilus. Brandt. Biological principles for the effects of ethanol. yeasts. Microbiol. Nakamura. 2.. K. Clark. B.S. Environ. Bengtsson. Jeffries. Biotechnol.Fuel Ethanol Production From Lignocellulosic Raw Materials Using Recombinant Yeasts 285 J. O. H. S. Li. J. cerevisiae. Genetic engineering of ethanol production in Escherichia coli. Y. 20. Appl.

402–408 (2005). . Pretorius and W. Lee and S.F. Expression of a Trichoderma reesei b-xylanase gene (XYN2) in Saccharomyces cerevisiae.A.C. 195 (2000). Expression of Aureobasidium pullulans xynA in.D. Kuyper. 38. van Dijken and J.G. Gonçalves and I. Hahn-H€gerdal.-F.J.S.T.J.S. 5. Hefner-Gravink and D. Lee. et al. 776–783 (1993). de Laat. Environ. FEMS Yeast Res.-F. Appl. 58.C. Appl. Y. P.W..A. Microbiol.. W. S. Environ. 968–972 (2004).J. Y. 1039–1046 (2007b). E. Hahn-H€gerdal. Microb. Claeyssens and W. Coa utilization of L-arabinose and D-xylose by laboratory and industrial Saccharomyces cerevisiae strains. P. Winkler. Biosci.. Microbiol.T. Environ. Appl. Kuyper. M.286 Ethanol and Butanol K. Microbiol. D. 62. High activity of xylose a reductase and xylitol dehydrogenase improves xylose fermentation by recombinant Saccharomyces cerevisiae. Gorwa-Grauslund. J. Kim. Saccharomyces cerevisiae. Wiedemann. M. 18. Leandro. Lighthelm. Fujita. I. Appl. Two glucose/xylose transporter genes from the yeast Candida intermedia: first molecular characterization of a yeast xylose-H þ symporter. M. Karhumaa. Garcia Sanchez. 4. Appl. 4. (2006). 543–549 (2006). Microbiol. P. J. Biochem. Ind. Spencer-Martins. 67. Environ. Karhumaa. Kondo. Mizuike. 62.M.. 63–68 (1988). K. A... Production of human caseino macropeptide in recombinant Saccharomyces cerevisiae and Pichia pastoris. Minimal metabolic engineering of Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: a proof of principle.C. W. I.T. Disruption of the yeast ATH1 gene confers better survival after dehydration. La Grange. Appl. Lindn. Hahn-H€gerdal. M. M... Cell Fact. 655–664 (2004).H. Microbiol. 32.P. Kuyper. Y. H. X. R. 73. Biotechnol.-J. Almering. 395. Appl. and ethanol shock: potential commercial applications. Kubota. Boles. 5407–5414 (2004). Karhumaa. Ljungdahl. A. Pretorius and W. Kim. Gorwa-Grauslund MF. La Grange.T. FEMS Yeast Res. 1036–1044 (1996) D. Harding. S. Jetten. Degradation of xylan to D-xylose by recombinant Saccharomyces cerevisiae coexpressing the Aspergillus niger b-xylosidase (xlnD) and the Trichoderma reesei xylanase II (xyn2) genes. E. B. H. Kim. A. Winkler. Pronk. A. Biotechnol.A. o Biotechnol. freezing. R. J. Hartog.P.H. J. 68. Alizadeh. Prior and J. Pronk. 186–191 (2002). 1661–1669 (1992). Stave. H. J. Gorwa-Grauslund MF. 54. T. K. Microb Cell Fact.S. Oh. FEMS Yeast Res. J. Isolation and characterization of acetic acid-tolerant e a galactose-fermenting strains of Saccharomyces cerevisiae from a spent sulfite liquor fermentation plant. B.H. L. M. Effect of ethanol on cell growth of budding yeast: Genes that are important for cell growth in the presence of ethanol.. Fukuda and A. W. B.E. M. Biotechnol. Metabolic engineering of a xylose-isomerase-expressing Saccharomyces cerevisiae strain for rapid anaerobic xylose fermentation. 69–78 (2003).. and secretion of the xylanase from. Fromanger. 1563–1569 (1996). T. J. J.. van Dijken and J. The oxygen requirements of yeasts for the fermentations of D-xylose and D-glucose to ethanol. Biotechnol. Appl.. Microbiol. Pretorius.. M. 70.. van Zyl. Appl. Claeyssens.-K. I. Microbiol. van Zyl. Microbiol.. Ryu. Xylose fermentation by Saccharomyces cerevisiae.J. 28.K. M. Microbiol. High-level functional expression of a fungal xylose isomerase: the key to efficient ethanolic fermentation of xylose by Saccharomyces cerevisiae?. du Preez. Kang. M. A. Bisson and J. K. Seo.J. Takeo. 6.. Toirkens. 399–409 (2005). Kinetic studies on glucose and xylose transport in Saccharomyces cerevisiae. 62. B. Appl. Pronk. van Zyl. Park..-L.J. Y. Environ. Construction of a xylan-fermenting yeast strain through codisplay of xylanolytic enzymes on the surface of xylose-utilizing Saccharomyces cerevisiae cells. Katahira.. Kume.H. Winkler. M. I. Hahn-H€gerdal and M.Y. B.C. M. Appl.J. Biotechnol. 5 (2007a).. Klionsky. Li and L. M. Biochem. K€tter and M. Op den Camp. Co-expression of the Trichoderma reesei xylanase 2 (XYN2) and the Bacillus pumilus b-xylosidase (xynB) genes in the yeast Saccharomyces cerevisiae.. M.A.R. Harhangi. D. La Grange. 5512–5519 (2001). 209–213 (1996).-F. Peetre and B. 60. A. Environ. Microbiol. Biotechnol. Microbiol.P.S. van Dijken and J. Microbiol. Comparison of the a xylose reductase-xylitol dehydrogenase and the xylose isomerase pathways for xylose fermentation by recombinant Saccharomyces cerevisiae. Ciriacy. den Ridder. 5.

Mishra. Chem. J.. W. S. Curr. Lett. Christensen. . Hahn-H€gerdal and G. Nissen.J. Kopp and H.. G. 197–237 (1998). Holzer. Hahn-H€gerdal. M. S. 24. Tolerance of fungi to ethanol. Nakari-Setala and M.R. Biotechnol. W.H. Pack. Penttil€. M. 10193–10198 (1995b). Appl. Metab. Xylose o a a isomerase activity influences xylose fermentation with recombinant Saccharomyces cerevisiae strains expressing mutated xylA from Thermus thermophilus. L€nn. W. Hahn-H€gerdal.P. T. 109–113 (1992). Yoo. Phenotypic features of trehalase mutants in Saccharomyces cerevisiae. Villadsen and M. L. E. I. 270. S.P. 286–290 (1995a).S. a Simultaneous saccharification and co-fermentation of glucose and xylose in steam-pretreated corn stover at high fiber content with Saccharomyces cerevisiae TMB3400. Moes.. Microbiol. Cloning of genes encoding a-Larabinofuranosidase and b-xylosidase from Trichoderma reesei by expression in Saccharomyces cerevisiae. 54. Yeast. E. Margolles-Clark. J. Arai and A. Appl.. Park and Y. Utilization of cellobiose by recombinant b-glucosidase-expressing strains of Saccharomyces cerevisiae: characterization and evaluation of the sufficiency of expression. J.. Ueda. Biochem.E. Gorwa-Grauslund. J. C. Biotechnol. Biotechnol. 4857–4861 (1998).. Secretion of a Cryptococcus albidus xylanase in Saccharomyces cerevisiae. Biol. Margolles-Clark. M. M. N.S. Mol. L. 116. K. Environ. M. Lynd. Nielsen. McBride. Biol. Appl. B. 360. 391–399 (1997). Enzyme Microb. Enhancement of b-glucosidase stability and cellobiose-usage using surface-engineered recombinant Saccharomyces cerevisiae in ethanol production.. Lynd. 411–415 (1997). Panagiotou. O. Christakopoulos. Meyer. 64. H.M. Biotechnol. Laser. 93–101 (2005). Marcel Dekker. Mechler. J. van Zyl and L. J. I.C. Fed-batch xylitol production with two recombinant Saccharoa myces cerevisiae strain expressing XYL1 at different levels. Luonteri and M. Nwaka. Opin. 126. Nwaka. Lett. Zacchi. Fukui and I. Morosoli.. Nwaka and H. Expression of a cytoplasmic transhydrogenase in Saccharomyces cerevisiae results in formation of 2-oxoglutarate due to depletion of the NADPH pool. Separate and o simultaneous enzymatic hydrolysis and fermentation of wheat hemicellulose with recombinant xylose utilizing Saccharomyces cerevisiae. B.L. 19. S€rensen. Nucleic Acids Res.S. J. Olsson.. D. T. I.-F.R. Progr. 104–111 (1996a).Fuel Ethanol Production From Lignocellulosic Raw Materials Using Recombinant Yeasts 287 A. J. Cloning of two b-xylanase-encoding genes from Aspergillus niger and their expression in Saccharomyces cerevisiae.R. 1919–1925 (2002). T. R. 18. Expression and function of the trehalase genes NTH1 and YBR0106 in Saccharomyces cerevisiae. van Zyl. Bengtsson. S. S. 488–498 (2006). A. New York. Holzer. Assimilation of cellooligosaccharides by a cell surface-engineered yeast expressing b-glucosidase and carboxymethylcellulase from Aspergillus aculeatus. M. S. Galbe. Öhgren.. Biotechnol Lett. Microbiol. T. M. Biochem. Tanaka. McBride and M.. van Zyl. Ohtsuki. Gene. Meinander and B. Tenkanen. 37.J. M. Technol. Bioeng.H. 54.L.J.. Molecular biology of trehalose and the trehalases in the yeast Saccharomyces cerevisiae. Cordero Otero. Environ. in: Stress Tolerance of Fungi. 19–32 (2001). Anderlund. Environ. T.. Krogh and A. M. 269–274 (1996). M. Machida. 240. 16. 8. 117–129 (2006). E.H.H. Microbiol.). van Zyl and B. (1993). P. Durand and R. Cloning and expression of the Clostridium thermosulfurogenes – xylose isomerase gene (xylA) in Saccharomyces cerevisiae. FEBS Lett. 3840–3846 (1996b). K. Engineering of the redox imbalance of Fusarium oxysporum enables anaerobic growth on xylose. Biotechnol.H. Moreau. 567–573 (2003). van Zyl. Tenkanen. Penttila. Pretorius and W. Appl. B. 32. H. Nucleotide sequences of Saccharomycopsis fibuligera genes for extracellular b-glucosidases as expressed in Saccharomyces cerevisiae. Technol.. 129–132.. Olsson. P. 18.. Enzyme Microb. K. Zietsman. Tr€ff-Bjerre. 474–482 (2006). Luttig.H. Destruelle and H. 62. 3147–3155 (1988). Holzer. Pretorius and W. Biotechnol. K.R. Yamashita. Three a-galactosidase genes of a Trichoderma reesei cloned by expression in yeast. Grotkjaer and L. Kielland-Brandt. Dam. 58. using glucose as a cosubstrate: a comparison of production parameters and strain stability. Jennings (ed. 577–583 (2005). Eng. Kawaguchi.. Murai.E. Consolidated bioprocessing of cellulosic biomass: an update. Eur. M.

H. Bioeng.S. 270–273 (1991). M. Sales. Andre. Investigation of the function of mutated cellulose-binding domains of Trichoderma reesei cellobiohydrolase-I. Nevanen.. Y. Ponaretou. D. Ruohonen. A. C. Boucherie.H. A. 140. Nakari-Set€l€. P. Environ. Saavedra.K. L. 4732–4736 (2003).. Saleh. Mol. Lindquist. Teleman and M. K. Stitzel and S. Genet. Microbiol. J.W. Construction of various mutants of xylose metabolizing enzymes for efficient conversion of biomass to ethanol. T. O. Lidn.M. Olsson. Shin. Hoffren. Regulation of mRNA export in response to stress in Saccharomyces cerevisiae. Microbiol. B. Rintala. Gonzalez-Villa and I. Rudolf.C. J.. Rumbak and G. Hopper and C.K. D.. Meacock. 103–112 (1988). Meacock. Gen. small endoglucanase a gene. Park. Raynal and J. Acta. 175–185 (1987). Lehtovaara and J.. Expression of the gene coding Aspergillus b-glucosidase. Saloheimo. glucose. Cloning and expression pattern of a gene encoding an a-xylosidase active against xyloglucan oligosaccharides from Arabidopsis. Teeri.S. Appl. 475–482 (1992). Production of ethanol from La arabinose by Saccharomyces cerevisiae containing a fungal L-arabinose pathway. S. Nucleic Acids Symp. 827–837 (2005). 121–127 (1995).E. 783–90 (2008). P. 249. K. Nature. Makino. 223. Watanabe. Verho. Funct. L. Brandt. A novel. B. Laaksonen.. The heat shock and ethanol stress responses of yeast exhibit extensive similarity and functional overlap. 126. Protein Struct. Pitk€nen. R. Hahn-H€gerdal and G. Biotechnol. Penttil€. Penttil€. G.A. Kim and M. Zacchi. (Oxf). Tung. 353. 80. Plant Physiol. 194. 99. Saloheimo. . J. T. A. Appl. M.T. C. P. including plasma membrane Hsp30. Richard. Genet. P.. M. M. Bailey.. Y. HSP12. Knowles. K. E. eg15. M. Simultaneous saccharification and a e fermentation of steam-pretreated bagasse using Saccharomyces cerevisiae TMB3400 and Pichia stipitis CBS6054.. Londesborough and M. 1463.K. P. Genet. Gen. Kwon. W. Xylose chemostat isolates of a a Saccharomyces cerevisiae show altered metabolite and enzyme levels compared with xylose. Penttil€. Nevalainen. P.D. A. Kraulis. Y. 35–44 (2000). 67. Knowles and T. 219–228 (1994).M. 494–499 (1984). Reinikainen. H. J. G. M.K. L. 185–189 (2003). Lehtovaara. 584–591 (1997). E.H. Penttil€. 1608–1620 (1996). Amberg. L. T. 279–280 (2006).A.E. J. Teeri and J. J. Jones. Penttil€. by ethanol level above a critical threshold. Penttil€. 50. Baudel.C. regulation and function. T. 69. The LEA-like protein HSP12 in Saccharomyces cerevisiae has a plasma membrane location and protects membranes against desiccation and ethanol-induced stress. 14. 13. T. Annaluru. Parsell. Kodaki and K. FEMS Yeast Res. Sieiro. M. Microbiol.P. K.. Penttil€.E. Piper.M. Induction of major heat-shock proteins of Saccharomyces cerevisiae. Eur.C.M. Tenkanen and M. Biotechnol..T. Lindsey. T. Biochem.M. Metabolic engineering of ammonium assimilation in xylosefermenting Saccharomyces cerevisiae improves ethanol production. Knowles. Sanchez. Genes Dev. Praekelt and P.K. M. Yang. Zarra. M. Moradas-Ferreira. Praekelt. Expression of two a Trichoderma reesei endoglucanases in the yeast Saccharomyces cerevisiae. cDNA cloning of a Trichoderma reesei aa a cellulose and demonstration of endoglucanase activity by expression in yeast. J. from Trichoderma reesei isolated by expression in yeast. Nielsen and L. J. T. T. Roca. D. Byrne. Putkonen. 3. Mol. A.W. Ser. P.. Biophys. Gene. 3. Henrissat. Revilla. Knowles.H. A. Ruohonen and M.C. Cole. 63. 134. 10. N. and ethanol metabolism of the original strain.288 Ethanol and Butanol E. Biochim. Lim. L.A. Saloheimo. Recnacq and H. P. Aristidou. 910–920 (2001). Mol.Y. 3031–3038 (1994). FEMS Microbiol. U. U. Efficient a secretion of two fungal cellobiohydrolases by Saccharomyces cerevisiae. 97–106 (1990). Andre. B. Lett. Expression of glucose oxidase by using recombinant yeast. Biotechnol.A. K. Sampedro. Talreja. A. 267–278 (2000). Piper. a new small heat shock gene of Saccharomyces cerevisiae: analysis of structure. Cloning of Aspergillus niger genes a in yeast.. Yeast. Hsp104 is a highly conserved protein with two essential nucleotide-binding sites. C. Microbiology.N..

D. 129–169 (2001). Sundstrom. J. Gonzalez-Candelas and D. G. K. 71–78 (1997). Biochem.J. 1615–1618. K. M. Environ. Environ. Sarthy.. T. 53. 113–116. Deletion of the GRE3 aldose a a reductase gene and its influence on xylose metabolism in recombinant strains of Saccharomyces cerevisiae expressing the xylA and XKS1 genes. T. M. Bioeng. Appl. Cloning and expression in Saccharomyces cerevisiae of a Trichoderma reesei b-mannanase gene containing a cellulose binding domain. coding for trehalose-6-phosphate synthase. Alcoholic fermentation of d-xylose by yeasts. Stanley.P. Cell Sci. Lindquist.. Biotechnol. Takahashi. . 1112–1119 (2001). Ramon. Microbiol. Furlong and B.R. 47. 53. J. Hahn-H€gerdal and H. 2357–2364 (1992). Pamment. Sanchez-Torres.A. Takemura. Biotechnol. 460–468 (1998). Arai. Appl. 4189–4197 (2004). Vehmaanpera. 3389–3394 (1990). M. 403–416 (2004). 1199–1204 (1993). K. Production of ethanol from cellulosic biomass hydrolysates using genetically engineered Saccharomyces yeast capable of cofermenting glucose and xylose. 21. B. Pamment. Douglas. Gacto. Hahn-H€gerdal. Ito. van den Bosch. Zacchi.Fuel Ethanol Production From Lignocellulosic Raw Materials Using Recombinant Yeasts 289 D. 2020–2024 (1999). Microbiol. Ademark. G.. 117. Appl. Lynd. C.J. Kawaguchi.  a H. Skoog and B. 1990–1998 (2003). Thermotolerance in Saccharomyces cerevisiae: the Yin and Yang of trehalose. 145. Environ.E. Singer and S.A. Soto. 99. Eleventh International Congress on Yeasts.E. Every. Stanley. J. Sedlak and N. H.H. Profiling the molecular response of Saccharomyces cerevisiae to ethanol stress using ethanol-tolerant mutants and gene arrays. A. Expression of the a Aspergillus aculeatus endo-b-1. Appl. Microbiol.D. 61. Shimoi and K.J. Accumulation of trehalose by overexpression of TPS1.. Takada. Toivola. E.4-mannanase encoding gene (man1) in Saccharomyces cerevisiae and characterization of the recombinant enzyme. Microbiol. M. M. Genomics.. Biores. W.V. Microbiol. A. 1090–1097 (1995).  P. Appl. Effect of acetaldehyde on Saccharomyces cerevisiae and Zymomonas mobilis subjected to environmental shocks. Sonderegger and U.. Biosci. Appl. W. Purif. D. 67.A. Cansado and M. in Proceedings. Sumitani and M. 5668–5674 (2001). J. T. Appl. Rogers and G. FEMS Microbiol. Stress response in yeast mRNA export factor: reversible changes in Rat8p localization are caused by ethanol stress but not heat shock. Stanley. Environ. J. 56. Lett. Brazil (2004).. Mol. Setati. 71. 16. Microbiol.. Expression in a wine yeast strain of the Aspergillus niger abfB gene.L. B.A.. 8069–8076 (2005). B. Microbiol. Genet. P.A. Biochem.R. Biotechnol. Evolutionary engineering of industrially important microbial phenotypes. Sauer. J.B. Hall. 1996–2000 (1987). Taulien. Martensson. Tzanatos and N. P. 265. Evolutionary engineering of Saccharomyces cerevisiae for anaerobic growth on xylose. Sanchez. G.. Izawa.. Tyurin. Chambers. Trends Biotechnol.B.. 62.. E. Technol. 11. Sullivan and L. 1221–1223 (1984). Biochem. causes increased resistance to multiple stresses in the fission yeast Schizosaccharomyces pombe. Borkovich and S. Stanley. P.A.. T. M.G. Otero Cordero. Henrissat and M. L. EMBO J. Inhibition and stimulation of yeast growth by acetaldehyde. 69. P. Hahn-H€gerdal. Effect of oxygenation on xylose fermentation by Pichia stipitis. A.W. Penttil€. Lobo. Steam pretreatment of H(2)SO(4)-impregnated Salix for the production of bioethanol. Protein Expr. 105–114 (2001). Sauer. 65. J. C. 137–145 (2008). Ho. Saloheimo. McConaughy. C.H. R. van Dijken and W. Role of spontaneous current oscillations during highefficiency electrotransformation of thermophilic anaerobes.V. N. Yarrow. U. Biotechnol. 189–194 (1996). van Zyl and B. van Zyl. Biotechnol. Inoue and S... S. a Appl. J. Microbiol. J. Expression of the Escherichia coli xylose isomerase gene in Saccharomyces cerevisiae. F-50 cellobiohydrolase I (cbhI) and b-glucosidase 1 (bgl1) genes by Saccharomyces cerevisiae. Lett. Vicente-Soler. Adv. Galbe and G. Fernandez.R. (1998). Environ. Lindquist. 73.A.G. 15. Fraser. Y. Scheffers. Environ. Environ. Appl.L. Identification of genes required for growth under ethanol stress using transposon mutagenesis in Saccharomyces cerevisiae. Tr€ff.. Z. Stalbrand. Eng.Y. R. Expression of Aspergillus aculeatus No. Hobley and N.. Sassner. Hsp104 is required for tolerance to many forms of stress.. T. Environ. Stalbrand.A.

E. Biotechnol.M. J. Clostridium phytofermentans sp.J.G. 14.B. London. Wahlbom and B. Jonson. S. Microbiol. W. 69.M. S. C. 23. Pretorius. J€nsson. van Zyl and I. Int. and acetoin act as external a electron acceptors during anaerobic fermentation of xylose in recombinant Saccharomyces cerevisiae. W. J.A. Kwok. L. Hahn-H€gerdal and R. Mol. Biotechnology.A. del Rosario Franco Berriel. Walfridsson.L. van Zyl. Leschine. characterization. Microbiol. 4184–4190 (1995).. 2043–2050 (2008). Van Dijken and J. Wang and H. den Haan and J. Planta and W. Otero. Ladner. Generation of the o a improved recombinant xylose-utilizing Saccharomyces cerevisiae TMB 3400 by random mutagenesis and physiological comparison with Pichia stipitis CBS 6054. W.. Wiedemann and E. Yeast. Furfural. B. van Voorst. Davies.R. Houghton-Larsen. H. P.A. R.H. J. 78. Lilius. Appl.. Eng. Biol. Environ. Engineering of Saccharomyces cerevisiae for efficient anaerobic alcoholic fermentation of L-arabinose. Londesborough. 319–326 (2003).W. 5892–5897 (2003). 62. J.R. J. Microbiol. Xylose-metabolizing a a a Saccharomyces cerevisiae strains overexpressing the TKL1 and TAL1 genes encoding the pentose phosphate pathway enzymes transketolase and transaldolase. M. J.F.S.A. X. and expression in Saccharomyces cerevisiae of endoglucanase I from Trichoderma reesei. Winkler.H. 61. Codon-optimized bacterial genes improve L-arabinose fermentation in recombinant Saccharomyces cerevisiae. 1165–1168 (1980). C. van Maris.H. M. F. Parekh. R. Yeast.Y. W. S. 30.S. Winkler. Innis. 60–64 (1987). Genome-wide identification of genes required for growth of Saccharomyces cerevisiae under ethanol stress. The value of electrophoretic fingerprinting and karyotyping in wine yeast breeding programmes..B.S.P. van Zyl and I.A. M. Brandt.. Meacock.H.P.S.. 6. Verho. Can. 67–76 (1998). van Rensburg. 3. 5. P. Wood hydrolysis by Cellulomonas fimi endoglucanase and exoglucanase coexpressed as secreted enzymes in Saccharomyces cerevisiae. FEMS Yeast Res. J. Hahn-H€gerdal. C. Warnick. W.H. Hallborn.. Engineering redox cofactor regeneration for a improved pentose fermentation in Saccharomyces cerevisiae.R.R. Biochem. Microbiol.J. Toirkens. Cell. B. Metabolic engineering of xylose-fermenting eukaryotic cells. Hahn-H€gerdal. De Laat. Microbiol. Consolidated bioprocessing for bioethanol production using Saccharomyces cerevisiae. G..A.T. 61. D. Construction of cellobiosea growing and fermenting Saccharomyces cerevisiae strains. Wisselink. van Dijken. Biotechnology. 205–235 (2007). 713–719 (1988). Kielland-Brandt and A. Wahlbom. which expresses an active xylose (glucose) isomerase. Environ. Skipper. Evol. D. Adv. Pretorius. L. Biotechnol. Appl. Walfridsson. van Zyl. Curr.C. 120. M. Curry. 5-hydroxymethyl furfural. Bao.H. 246–250 (1996). 74. L. Hahn-H€gerdal.. R. Syst. L. Gelfand and M. Engineering yeast for efficient cellulose degradation. Ethanolic u a fermentation of xylose with Saccharomyces cerevisiae harboring the Thermus thermophilus xylA gene. M. van Rensburg. Co-expression of a Phanerochaete chrysosporium cellobiohydrolase gene and a Butyrivibrio fibrisolvens endo-b-1. Growth of yeasts on D-xylulose 1.. J.W. D. Varela. van Zyl. 108.4-glucanase gene in Saccharomyces cerevisiae. John Wiley and Sons. Pronk. W. 4648–4651 (1996). A. S. . Yeast Physiology and Biotechnology.F. Kuyper. van Rooyen.H. 73. 284–295 (2005). J.J.J. Environ. Methe and S. Antonie Van Leeuwenhoek. J. A. U. Schweickart. Pretorius.. Ker€nen. Boles. R. McBride. M. Richard. 172–178 (2002).290 Ethanol and Butanol J.M. Microbiol. Appl. 4881–4891 (2007). Schneider. 52. Genet. Appl.N. 1988. La Grange and W. Microbiol. T. M. Biotechnol. Penttil€. 249–257 (1992). Walker. 15.M. a cellulolytic mesophile from forest soil. Lynd.J.C. Mager.... van der Westhuizen and I. van Arsdell. nov.A. Pronk and A. P. 26. R. 351–359 (2006). Kilburn and N. Wong.K. B. B. B€low and B. Bioeng. 1155–1160 (2002). Penttil€ and P.T. Hahn-H€gerdal. P. Appl. R. The Saccharomyces cerevisiae HSP12 gene is activated by the high-osmolarity glycerol pathway and negatively regulated by protein kinase A. International patent application WO 2006/009434A1 (2006). T. Cloning. G. Anderlund.T. B. M. V. Parekh. M. Environ. Praekelt. M. 6232–6245 (1995).C. Wayman. Environ.

strain PC-2 has carbohydrate binding and docking modules. Ramon and J. 321. 267. Zhang. Construction of an endoglucanolytic brewing yeast strain. 101. 240–243 (1995). Picataggio. J. Updegraff.Fuel Ethanol Production From Lignocellulosic Raw Materials Using Recombinant Yeasts 291 J. Deanda. Li. Cotta. Ljungdahl and X. Wu. Eddy. Xie. Can. S. Perez-Gonzalez. D.A. H.A. M. M. Felix.G.F. 459–461 (1995).. Nature. ManA. E. K. 51. Metabolic engineering of a pentose metabolism pathway in ethanologenic Zymomonas mobilis. Finkelstein and S. Q. A mannanase. Jimenez. Ethanol production from sugars derived from plant biomass by a novel fungus..M. C. Lastick and D. M. Chem. of the polycentric anaerobic fungus Orpinomyces sp.A. J. .L. I.R. Ximenes. Kataeva.A. Inst. Science. C. L. Microbiol.M. 887–888 (1986). 559–568 (2005). Brew. A.


ethanolrfa. . With a projected increase of flexible fuel vehicles which operate on gasoline containing up to 85% ethanol (E85).epa. with the implementation of the first Renewable Fuels Standard in 2007 (http://www. the US consumed approximately 140 BG of gasoline in 2004 (http://www. has been widely used as a transportation fuel in the Similarly.8 billion gallons (BG) in 2006 (http://www. Furthermore. the domestic production of ethanol is far behind the demand for transportation liquid fuel.. the extensive use of corn starch for ethanol production would result in a competition for feedstock. Nasib Qureshi. which contains 90% or higher gasoline blended with 10% (E10) or lower ethanol (E5).doe. According to the annual energy outlook. and thus result in economic stress in the food and feed industry. Gasohol. 1977). Hans Blaschek e and Hideaki Yukawa The contribution of Liu has been written in the course of his official duties as US government employee and is classified as a US Government Work.eia. and extensive research in the area of pretreatment and fermentation has been carried out Biomass to Biofuels: Strategies for Global Industries Edited by Alain Verts.14 Conversion of Biomass to Ethanol by Other Organisms Siqing Liu 14. The needs to use lignocellulosic biomass materials as alternative renewable feedstocks for ethanol production were recognized during the early 1970s (Wang et al.1 Introduction Sugarcane-based bioethanol has been the major automobile fuel in Brazil for almost three decades due to the abundance and renewable nature of sugarcane in that country. which is in the public domain in the United States of America. corn starch-based ethanol production in the US has expanded from 175 million gallons in 1980 to

including: (i) the efficient transportation of bulky biomass feedstocks to conversion sites. 1988).. However. rather than glucose alone. significant breakthroughs have been achieved. in the presence of inhibitors. and (iv) a lack of practical and cost-effective processes to reduce waste stream and increase product recovery efficiency. In this chapter. is present in the biomass hydrolysates (Wright et al. . there are biological limitations of available biocatalysts. (iii) a lack of efficient biocatalysts and optimal processes for converting mixed sugars derived from lignocellulosic biomass into ethanol and valuable coproducts. 2007). Recent successes in developing eukaryotic microbes for biomass conversion have been reviewed elsewhere (Jeffries and Jin. Han et al. and to continue to perform these functions with an increased ethanol content. Although tremendous efforts have been made to meet these challenges.294 Ethanol and Butanol over the subsequent four decades (Flickinger. 1980. attention is focused on the current status in developing prokaryotic organisms for achieving the cost-effective conversion of biomass to biofuels and other value-added products. The microbes are desired to ferment sugar mixtures with a high ethanol yield and productivity. van Maris et al. However. 2003. In addition.. while microbial strain development remains one of the key success factors for the lignocellulosic biomass-to-ethanol industry. Saha. 2002.2 Desired Biocatalysts for Biomass to Bioethanol Conventional Saccharomyces for starch-to-ethanol fermentation is not applicable for lignocellulose-based ethanol production. (iii) the efficient fermentation of the mixed sugars present in biomass hydrolysates into ethanol. various fermentation inhibitors are released or formed during the pretreatment and hydrolysis steps of lignocellulosic biomass feedstocks (see Chapter 12).. additional research is required towards strain development in order to achieve the economic conversion of biomass to fuels and chemicals. Sun and Cheng. as a mixture of sugars and oligosaccharides. The primary challenge in developing robust microbes is to ensure the efficient conversion of concentrated and mixed sugars and oligosaccharides from biomass hydrolysates into ethanol. As a result. Major barriers for the cost-effective production of ethanol from lignocellulosic biomass feedstocks include both logistical and technical constraints.iogen. pathways for producing endogenous hydrolytic enzymes must be engineered in fermenting microbes so as to accommodate (ideally) a cost-saving single bioreactor configuration (Lynd et al. 1993. 2002). (ii) costly processes and hydrolytic enzymes to breakdown heterogeneous and recalcitrant lignocellulosic biomass into simple fermentable sugars. Current ‘biomass-to-ethanol’ technologies have demonstrated success in pilot-plant production and feasibility in commercial scale-up (http://www. The root causes associated with these barriers are: (i) variations of biomass feedstocks at different geographic locations and the relative low density of lignocellulosic feedstocks. Lawford and Rousseau. (ii) an efficient degradation of biomass materials into hydrolysates containing fermentable sugars. 1981. 2003). 14. and (iv) cost-effective downstream processes for recovery of ethanol and byproducts.. the process designs for achieving the conversion of biomass to ethanol are more complicated and costly than the simpler starch-to-ethanol processes. and also described in Chapter 13 of this book. Technological breakthroughs in all these areas are essential for the overall economics of the emerging biofuel value chain.

and xylB) and the pentose phosphate pathway (talB and tktA) are shown in bold italics. The contaminated growth of this microbe in cider led to increased turbidity.3 14. PPP ¼ pentose phosphate pathway .1 Gram-Negative Bacteria Zymomonas mobilis This Gram-negative bacterium was first isolated in 1911 by Barker and Hillier from cider sickness (Swings and De Ley. mobilis AX101.3-P-Glycerate 3-P-Glycerate 2-P-Glycerate Phosphoenolpyruvate Lactate ED Pyruvate Pyruvate Decarboxylase Pyruvate Acetyl-CoA Acetate Acetaldehyde Alcohol Dehydrogenase Ethanol Figure 14. In Z. glucose is metabolized via 2-keto-3-deoxy-6-phosphogluconate aldolase (KDPG aldolase). These observations suggested the occurrence of a typical microbial sugar-to-ethanol fermentation. araB. xylA. araD.1). Key enzymes unique for Z mobilis are highlighted in a box with black background. an altered aroma and a reduced sweetness. mobilis. The introduced foreign genes for pentose assimilation (araA.3.Conversion of Biomass to Ethanol by Other Organisms 295 14. The pathways are labeled with abbreviated capital letters. one of the key enzymes in the Entner–Doudoroff (ED) pathway (Figure 14. ED ¼ Entner–Doudoroff pathway. The outcome of this reaction is the Sucrose Glucose Glucose-6-P Fructose Fructose-6-P Arabinose araA Ribulose araB Gluconolactone-6-P Gluconate-6-P 2-keto-3-deoxy-6-P-Gluconate Ribulose-5-P araD Xylose xylA Xylulose xylB Xylulose-5-P Fructose-6-P Sedoheptulose-7-P Xylulose-5-P + Erythrose-4-P talB + tktA Ribose-5-P + Ribose-5-P Glyceraldehyde-3-P KDPG Aldolase PPP Glyceraldehyde-3-P 1.1 Schematic diagram of mixed sugar metabolism in engineered Z. 1977). accompanied by a significant production of ethanol and CO2.

1979) At least two pilot-scale processes. a chromosomal-integrant derivative of ZM4:pZB5..1) and pentose phosphate pathway genes (tktA and talB. Z. as compared to yeast. mobilis ATCC 10988 (Zhang et al. and hence the commercial risk associated with plant trials in a tight production campaign schedule. Another critical factor appears to be the lack of experience in industrial-scale fermentations using Z. producing ethanol at 97% of the theoretical yield and a relatively low bacterial biomass accumulation (Rogers et al.. 1993). Comparative batch fermentations (Lawford et al. 2001). This was constructed by fusing the E. mobilis for ethanol production.296 Ethanol and Butanol production of ethanol at high efficiency (Seo et al. mobilis. genetic engineering approaches have been used to broaden the substrate range of Z. Figure 14. These processes were tested with sugarcane syrup. the sensitivity of this microbe to acetic acid levels. Moreover. mobilis was considered to be the fastest-acting ethanologen from glucose.. pZB5. mobilis 8b. Z. molasses (Doelle et al.. Noteworthily. increased ethanol contents. Despite this. 1995b). as it exhibited not only a better yield and productivity but also had an increased tolerance towards ethanol and acetic acid. 2005). low pH. Figure 14. beet molasses..’ were developed using Z.. mobilis. coli plasmid pACYC184 to a 2. However. Likewise. One reason for this lack of translation of an apparently useful academic discovery into the bio-industry could be the relatively low bacterial cell mass production attained by this bacterium. and bacterial contamination might also have negatively contributed to its commercial application into the starch-to-ethanol industry. mobilis metabolizes a narrow range of hexoses. milo. 1996). this . mobilis CP4 (pZB5) was first developed by introducing xylose assimilation (xylA and xylB.1) from Escherichia coli via a shuttle vector. arabinose metabolism genes (araB. This trait becomes a key limiting factor for direct applications in lignocellulosic biomass conversion.. and sago (Doelle et al. the pZB5 plasmid carrying the xylose-utilizing pathway genes was introduced into strain ZM4 to construct strain ZM4:pZB5. almost all currently available commercial ethanol plants use Saccharomyces species rather than Z.. the resultant strain C25 showed a greater than 80% yield from glucose and xylose mixtures. 1987) and the ‘Glucotech process. Approximately 86% and 95% of the theoretical yields have been achieved with xylose alone or glucose and xylose mixed fermentations using the resulting strain (Zhang et al. 2001) of oat hull hydrolysates supplemented with corn steep liquor using recombinant strains and one strain adapted to hardwood prehydrolysate fermentation indicated that ZM4:pZB5 could outperform all of the other Z. 2004). Similar to conventional Saccharomyces. as this would add extra expense for the preparation and delivery of starter cultures. However. mobilis strains tested.1) and pentose phosphate pathway genes (tktA and talB.1) were assembled in plasmid pZB206 (Deanda et al. and also an increased sensitivity to acetic acid (Lawford and Rousseau. 1995a). the naturally occurring Z. 1991). For example. potato. corn starch. Z. cassava. During the late 1970s. yielded 85% ethanol from corn stover hydrolysates produced by dilute acid pretreatment (Mohagheghi et al.. maltrin.. but cannot utilize pentoses such as xylose and arabinose – two carbohydrates that constitute the major sugar pools released from biomass hydrolysis (Zhang et al. Figure 14. 1995). including the multistage ‘Bio-Hol process’ (Lawford. araA. and araD. Similarly. mobilis by introducing genes required for pentose metabolism.7 Kb plasmid from Z. This stability of the pentose-utilizing trait was later improved via chromosomal integration of the plasmid-carrying transgenes. mobilis strain harboring pZB206 produces ethanol at near-theoretical yield using glucose and arabinose. The recombinant Z. also known as strain 31821:pZB5. Figure 14. to develop a process for converting lignocellulosic feedstocks into fuel ethanol using genetically modified strains of Z. see Figure 14. mobilis 206C (pZB301) the ability to coferment glucose. retained the capacity to utilize xylose and arabinose after 160 generations in nonselective media. the genes for xylose/arabinose assimilation and the pentose phosphate pathway (a total of seven genes. mobilis recombinant strains are still among the most promising biocatalysts for the biomass-to-ethanol industry. This is perhaps best exemplified by the partnership recently announced by the companies Dupont (Wilmington.. and E. 2007.. xylose. in this strain. Z. DE. it was possible to eliminate the antibiotic selection by generating stable chromosomal integrants using either targeted homologous recombination or random mini-Tn 5 and Tn 10 systems (Mohagheghi et al. that conferred to Z. with the glucose pool being used first. 1993). http://www. one out of the 12 resulting strains. this preconditioning process unfortunately has certain drawbacks. mobilis strain AX101 has been observed to outperform an industrial yeast strain in terms of both conversion efficiency and yield (Lawford and Rousseau. the lactate dehydrogenase gene had been replaced by arabinose utilization genes. US) and Broin (renamed as Poet.. US. an overliming step has been proposed to adjust the pH and reduce acetic acid content (Mohagheghi et al. including the fact that is often leads to an accumulation of gypsum which in turn typically results in increased operating costs due to filtration requirements. 14. 2006). facultative anaerobic bacterium was discovered by and named after Theodor Escherich in 1885. and arabinose (Zhang et al. mobilis (Doelle et al. followed by those of xylose and arabinose (Mohagheghi et al. 2002). the major obstacle to the industrial implementation of AX101 has been its low tolerance to acetic acid. mobilis.. Nevertheless.html). coli has been a model system for . when acetic acid reaches levels above 2. in turn.3. Z. the rate of pentose utilization slows down by 50%.2 Escherichia coli This Gram-negative.5 g lÀ1 (pH 5. sorbitol. Although successful. Sioux Falls. Batch fermentation tests with simulated biomass hydrolysates suggested that strain AX101 could ferment mixed sugars to ethanol with better yields when compared to strain C25. Moreover. Specifically. the strain showed a preferential order of utilization. 2003). AX101 produced less lactic acid because.5). Clearly.. or fructo-oligosaccharides by Z. 2006/10/dupont_and_broi. mobilis (Rogers et al. are important and will potentially reduce the cost for ethanol production using genetically engineered Z. 1998). Additional research exploring the production of other value-added products such as levan. Furthermore.. this was an important observation as this trait decreased the overall industrial robustness of the strain. In order to use lignocellulosic biomass hydrolysates in AX101fermentations. Moreover. mobilis AX101. However. the strain also showed an increased sensitivity to acetic acid. SD. In the Separate Hydrolysis and Fermentation process (SHF)..poetenergy. pZB301. Remarkably.1) were combined in a single plasmid. and to coferment all three sugars with a yield of 84%. 2002). 1996). 2002). this leads to a dramatically decreased ethanol yield and productivity (Lawford and Rousseau. However.Conversion of Biomass to Ethanol by Other Organisms 297 plasmid-bearing strain has been observed to lose its ability to ferment arabinose within seven generations when grown in the absence of a positive selected pressure (such as the presence of an antibiotic in the fermentation medium) (Deanda et al. the engineered Z. as well as novel biomass degradation processes that reduce acetate levels in biomass hydrolysates.

The resulting recombinant strains include E. mobilis were cloned and introduced into E. The assembled ethanol production pathway including pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) genes. coli for lignocellulosic biomass-to-biofuel applications is its natural capacity to metabolize pentoses. lactate. 2000).2) of E. 1949. coli. and molecular biology ever since (Stokes. coli K-12 TC4 transformed this low-ethanol-producing bacterium into one that produces ethanol as its main fermentation product (Ingram et al. Clark.2) (Neidhardt et al.2). 1989). this phenomenon constitutes an adaptation strategy implemented by this organism to optimally cope with varying environmental conditions. Some of these recombinant organisms are among the most promising biocatalysts in the lignocellulosic biomass-to-ethanol fermentation industry (Antoni et al.. 1987). No studies of this microbe were associated with large-scale ethanol production until after 1987. These studies constituted an important turning point. In addition. 1991). coli strains have subsequently been generated. DC1368 FBR4 (pLOI297). Meanwhile. coli strains to improve plasmid stability and ethanol production. 1991). This plasmid was later introduced into other E.. was initially assembled in a replicative plasmid. The intermediate metabolite pyruvate is then fermented into organic acids (including lactic.. and reduced acetate. About 95% of cells maintain the plasmid when cultures are serially transferred 10 times under anaerobic conditions. 1989). 2007). mobilis into E.. and succinate production . coli ATCC 9637 (pLOI297). Fliege et al. better productivity. The most attractive feature of E. coli ATCC11303 to generate strain KO11 (Ohta et al. ATCC 15224(pLOI297) (Alterthum and Ingram. comparisons of pH-controlled glucose or xylose fermentation indicated that strain KO11 exhibits a higher ethanol yield. coli ATCC11303 (Ohta et al. coli using recombinant DNA technology (Ingram et al. the recA mutation was introduced to minimize recombination instability.2) was introduced by conjugation of the Tn10-based E coli K-12 frd mutant SE1706 into E. 2003). Moreover. FBR2 (Hespell et al. 1989). and a series of recombinant E. increased cell yield. a mutation of the fumarate reductase gene (frd) (Figure 14. Hexoses are catabolized via both the ED pathway and the Embden–Meyerhof pathway (EMP) into pyruvate (Figure 14. the stable chromosomal integrants KO1–KO5 and KO20 were generated by targeted insertion of the pet operon at the pfl locus (Figure 14. resulting in strains KO10 and KO12 (Ohta et al. coli was reported during the 1940s. 1989) Recombinant DNA technology that enabled the introduction of the ethanol pathway from Z.. 1991). acetic. when the observed predominant fermentation products were identified as organic acids (Stokes.. Furthermore.. and NZN111 FBR5 (pLOI297) (Dien et al. 2000). The nature and ratio of the final fermentation products vary with the availability of fermentation substrates and the pH of the culture medium. the fermentation of corn fiber hydrolysate by FBR5 reaches 90% of the theoretical ethanol obtainable under anaerobic conditions (Dien et al.. 1987). 1990. this adaptation is achieved through balancing the number of reducing equivalents generated by each pathway (Clark. Among these. 1996). FMJ39 FBR1. Anaerobic fermentation by E. when the ethanol-production genes from Z. these sugars enter the carbon metabolic pathways via the pentose phosphate pathway (PPP) (Figure 14.298 Ethanol and Butanol bacterial physiology. Essentially. biochemistry. Overall. pLOI297 (Alterthum and Ingram. strain FBR5 shows an improved stability of plasmid pLOI297 in the absence of antibiotic-mediated selective pressure. named the pet (Production of EThanol) operon. 1992. ATCC 11303(pLOI297). and succinic acids) and into small amounts of ethanol via branched pathways (Figure 14.. Hong et al. 1949).. In E.2)... formic. genetics.

Three carbohydrate metabolic pathways are illustrated and labeled with abbreviated capital letters.Conversion of Biomass to Ethanol by Other Organisms Glucose Glucose-6-P Fructose Fructose-6-P Phosphofructokinase Gluconolactone-6-P 299 Arabinose. and waste house wood (Okuda et al. ..6-P Fructose-6-P Sedoheptulose-7-P Xylulose-5-P EMP Gluconate-6-P + Erythrose-4-P talB + tktA + Ribose-5-P Glyceraldehyde-3-P Aldolase 2-keto-3-deoxy-6-P-Gluconate PPP Dihydroxyacetone-P KDPG Aldolase Glyceraldehyde-3-P ED 1. EMP ¼ Embden–Meyerhof–Parnass pathway (Ohta et al. the ethanologenic E.. mobilis are shown in bold italics.. 2000). 1998).2 Schematic diagram of mixed sugar fermentation in engineered E. orange peel (Grohmann et al. this simple technique of metabolic evolution proved to be successful. Notably. coli KO11. Xylose Xylulose-5-P Fructose-1. Remarkably. corn stover. 1991).. The introduced ethanol-production genes pdc and adh from Z. sugarcane bagasse (Asghari et al.3-P-Glycerate 3-P-Glycerate 2-P-Glycerate Phosphoenolpyruvate Lactate ldh Fumarate pfl frd Succinate Pyruvate Formate Acetate ack Acetyl-P Citrate pta Acetyl-CoA adh pdc Acetaldehyde adh Acetaldehyde adh Ethanol Ethanol Figure 14. Key enzymes for the carbohydrate metabolic pathway are highlighted in a box with a black background.. coli KO11 was subjected to extensive fermentation studies with various biomass hydrolysates including corn fiber. Furthermore. 1996). 2007). the ethanol-tolerant strain LY01 was developed based on strain KO11 by using long-term adaptation on medium supplemented with increased ethanol contents. since the ethanol tolerance of strain LY01 was seen to be 10% higher than that of the parent KO11 strain (Yomano et al... beet pulp (Doran et al. softwood (Barbosa et al. 1996). 1992).

com/)..4 Gram-Positive Bacteria Gram-positive bacteria possess several desirable traits for the production of bioethanol. First. that started operation in June 2007 (Verenium Corporation: http://www. 1992).6. most LAB species are ethanol-tolerant. 2007). 2007). 2005) by transposonmediated mutagenesis and metabolic evolution (Jarboe et al.1 Lactic Acid Bacteria Lactic acid bacteria (LAB) lack cytochromes and are obligate fermentative organisms.html). temperature 37  C) and the hydrolytic enzymes (typical optima are pH 4. are known to grow in the presence of inhibitors derived from plant materials . com/2006/08/celunols-wet-biomass-conversion.. and glucose fermentation are all performed in separate tanks (http://bioconversion. cellulose hydrolysis. This strain was reportedly used in a demonstration plant in Jennings. temperature 55  C). Functional selections were subsequently performed by serial transfers in mineral salts medium without antibiotics (Zhou et al. Like other biomass-to-ethanol Separate Hydrolysis and Fermentation (SHF) designs. adhA. some strains exhibit relatively high process robustness as they are relatively tolerant to environmental stresses and are capable of growth at a relatively large temperature range (30–50  C) (Bothast et al. given the temperature and pH incompatibility of the fermenting microbe (typical optima are pH 6.. the relevant fermentation technology was licensed to Marubeni Corporation for constructing and operating in Osaka... xylose fermentation. A combination of genetic engineering and long-term adaptation has resulted in microbial biocatalysts that produce up to 45 g ethanol per liter in 48 h in a simple mineral salts medium.blogspot. Some utilize various carbohydrates.. Nevertheless. 2007). This group of microbes should be explored more extensively for potential applications in converting biomass to biofuel and chemicals (Verts et al. which was engineered for ethanol production from the lactic acid-producing strain SZ110 (Zhou et al.verenium. In addition. Verenium Corporation announced the plans to build its first commercial-scale cellulosic ethanol facility in Highlands County. the world’s first commercial-scale cellulosic ethanol plant using woody biomass (Antoni et al. Japan.4. LA.300 Ethanol and Butanol An interesting construct is that of strain LY168. The strain LY168 is among the best engineered E. Florida. such as Lactobacillus brevis and Lactobacillus buchneri. 1999). Certain species.5. 14. hemicellulose hydrolysis. Recent research using Gram-positive bacteria as biocae talysts for biomass-to-biofuels are discussed below. 2009). This constraint makes the ‘Celunol Ethanol from Biomass Process’ expensive to operate. More recently (15 January. 2005). including pentoses/hexoses and sugar derivatives for energy production via substrate-phosphorylation. US (http://www.. including the ability to ferment multiple sugars simultaneously and to grow at lower pH-values. substrate the processes for pretreatment. Moreover. 14. and capable of growing in media containing up to 10–16% of ethanol (Gold et al.verenium. 2008). coli strains for the production of ethanol from xylose in mineral salts media supplemented with betaine (Jarboe et al. the transposon containing promoterless pdc.. and adhB genes was randomly inserted into the chromosome of SZ110.

1967). 2001) into Lactobacillus plantarum TF103 (a strain in which both the ldhL and ldhD genes were inactivated) resulted in a recombinant strain that produced up to 6 g lÀ1 of ethanol from 40 g lÀ1 glucose (Liu et al. Sakamoto and Konings. ventriculi pdc was also introduced into Bacillus megaterium (Talarico et al. reuteri NRRL B-30867. Most LAB can grow and ferment in acidic environments (De Angelis et al. 1999... 2007).. producing 0. as these requirements are compatible with the optimal pH for biomass hydrolytic enzymes (Ohgren et al.3) (Keenan and Lindsay. in engineered LAB strains. it was anticipated that introduction of the PDC gene would transform targeted LAB strains into ideal ethanologens (Figure 14. 2003). resulting in ethanol production at varied levels. and L. in which ethanol is produced by PDC and ADH in a stepwise decarboxylation and dehydrogenation process. Consequently. It is worth noting that LAB have been used in the dairy industry to produce an array of value-added compounds including nutraceuticals. 1987) into Lactobacillus casei (Gold et al. The introduction of the pdc gene from Sarcina ventriculi (Talarico et al.7 g lÀ1 and 6.. This suggested that the endogenous ADH in L. 2002. 1999). cerevisiae and Z. However. mobilis. crispatus NRRL B-30868.. and alcohol dehydrogenase (ADH) (Figure 14.. buchneri PTA6138.9 g lÀ1 ethanol from 5 g lÀ1 xylose. lactis-specific promoters (De Ruyter et al. 1997) and the CP40 promoter from Lactococcus lactis (Jensen and Hammer. and on the other hand of naturally producing approximately 22 g lÀ1 ethanol from 60 g lÀ1 glucose (Liu et al..Conversion of Biomass to Ethanol by Other Organisms 301 such as wine polyphenolics or hop acids that are typically present in beer (LonvaudFunel. lactic acid. casei and recombinant PDC are sufficient for achieving efficient ethanol production. bacteriocins. This is an important property in view of industrial applications. 2006). 2003). NRRL B-30866. ventriculi pdc nor a construct adding an adh gene increased the ethanol production in L. Selected LAB including L... The potential production of value-added products from biomass materials paralleled with ethanol fermentation could further reduce the overall cost for biomass to ethanol production. despite this strain being capable on the one hand of fermenting both xylose and glucose. acetaldehyde dehydrogenase. These studies indicate that. brevis NRRL B-30865. Since the genomes of LAB bear ADH but not PDC. 2003). Increased acetaldehyde concentrations were observed when the pdc gene from either Z. 30870. 1998). neither a construct adding the S. sorbitol. Calderon Santoyo et al. 2006).. Unlike S.. 30869. and mannitol (Hugenholtz et al.. can produce ferrulate esterases.. 2001. the heterofermentative LAB produce ethanol via pyruvate dehydrogenase. brevis ATCC367. 2006). mobilis (Ingram et al... the pet operon was introduced into other lactic acid bacteria under the control of the ldh promoter (Nichols et al. 2005). 2005.. Madsen et al. thereby suggesting that a functional expression of pdc could be achieved under the control of nisA and other L.. Recombinant strains expressing the pdc gene or the entire pet operon respectively produced 8. 1996. 1996) using the Bacillus subtilis SPO2 promoter.5 g lÀ1 ethanol from 40 g lÀ1 glucose. 2005). lactis (Bongers et al. Liu et al. Kleerebezem and Hugenholtz. research was carried out to introduce the pet operon including the pdc and adhB genes from Z.3). 2003). Similarly. depending on the host species examined (Nichols et al. mobilis or Zymobacter palmae was introduced into L.. L. The S. the redirection of carbon flow from . these enzymes may have an unique role in lignocellulosic biomass hydrolysis as they break down the crosslinks between lignin and hemicellulose (Nsereko et al. 2003) from Streptococcus bovis (Wyckoff et al. L.

It is apparent that a different strategy is needed to improve the efficiency of ethanol production using LAB.6-P Aldolase Phosphoketolase Glyceraldehyde-3-P 1.3 Schematic diagram of mixed sugar fermentation in heterofermentative lactic acid bacteria. ethanol is produced naturally via acetaldehyde from acetyl-CoA. (ii) the addition of exogenous pyruvate decarboxylase and alcohol dehydrogenase. Key enzymes for the carbohydrate metabolic pathway are highlighted in a box with black background. and energy requirements need also to be considered in designing new engineering approaches. a genetic knockout system for pentose-utilizing LAB strains must be developed. Other factors including redox balance. pentoses can also be catabolized via the pentose phosphoketolase pathway (PKP) lactate to ethanol is not easily achieved due to competition for pyruvate between lactate dehydrogenase and pyruvate decarboxylase. buchneri and L.3-P-Glycerate 3-P-Glycerate pta PKP Dihydroxyacetone-P EMP Acetyl-P ack 2-P-Glycerate Phosphoenolpyruvate Acetyl-CoA Pyruvate ldh pdc adh Acetate Acetaldehyde adh Lactate Ethanol Figure 14. In heterofermentative LAB. . NADH/NAD þ ratio. (iii) supplementation of acetylCoA precursors to enhance carbon flow from acetyl-CoA to ethanol via acetoaldehyde.302 Ethanol and Butanol Glucose Fructose-6-P Glucose-6-P Gluconate-6-P Arabinose Ribose Ribulose-5-P Ribose-5-P Xylose Xylulose Xylulose-5-P Phosphofructokinase Fructose-6-P Sedoheptulose-7-P Ribose-5-P + Erythrose-4-P + Glyceraldehyde-3-P + Xylulose-5-P PPP Fructose-1. brevis. Engineering for ethanol production would encompass the following strategies simultaneously or sequentially: (i) inactivation of lactate dehydrogenase and acetate kinase steps to prevent carbon flow to lactate and acetate respectively. First. In xylose-fermenting LAB such as L.

. glutamicum (Inui et al. succinic acid.4. ventriculi has a lower affinity to pyruvate (Talarico et al. acetate. 2003. buchneri strain NRRL B-30929 was identified that grows rapidly in the presence of high concentrations of xylose (Liu et al. 2005).caltech. This strain produces 12 g lÀ1 of ethanol from 125 g lÀ1 mixed sugars. in addition to lactate. 7 g lÀ1) under oxygen-deprivation conditions in the host strain carrying the ldhA (lactate dehydrogenase) mutation. removing the undesired pathway that leads to acetate production (Figure 14.jgi. This strain was selected for genome sequencing by the Department of Energy (http://www. However.3)... 2006).. Another strategy would be to replace the endogenous lactate dehydrogenase with a heterologous PDC. The engineered strain produces threefold more ethanol (ca.doe. . and oligosaccharides. the addition to the culture media of pantothenate (the immediate precursor of CoA) resulted in increased ethanol fermentation (Richter et al. Strains from this particular group have been used at the commercial scale to produce proteinases as well as other enzymes and proteins.Conversion of Biomass to Ethanol by Other Organisms 303 hence. 2008). a group of bacterial isolates from a commercial ethanol plant were examined and a L. and utilizes a broad spectrum of DOEmicrobes2007.2 Corynebacterium glutamicum Another Gram-positive species that was explored for ethanol fermentation is Corynebacterium glutamicum. glucose-mediated regulation is still exerted on xylose consumption.) through the Joint Genome Institute.. with the resultant strain producing lactic acid. In a fermentation study using Oenococcus oeni. in which the Z. brevis (Makarova et al. buchneri. glutamicum was engineered to include xylA (xylose isomerase) and xylB (xylulose kinase) genes from E. Since the only available Gram-positive PDC from S. Genome comparisons of the L. coli (Kawaguchi et al. if the pool of acetyl-CoA is increased the carbon flow to acetaldehyde might be – is necessary to obtain a more favorable PDC with higher pyruvate affinity and increased expression/stability in Gram-positive hosts.. a genetic engineering approach – perhaps via directed evolution techniques (http://www. 14. including various monosaccharides (pentoses and hexoses). and acetic acid from xylose. would be critical in developing efficient LAB for fuel production. 2004).4.html. che. or converting acetate to other valuable byproducts. subtilis is another organism that has GRAS status (Generally Recognized as Safe) and is capable of utilizing a wide range of sugars. Wagner et al. disaccharides. 2008).3). Phenotype microarray and pH-controlled fermentations have suggested that xylose can be metabolized via the PPP and/or the phosphoketolase pathway (PKP) (Figure 14. mobilis pet operon was placed under the ldhA promoter from C. L. In a search for naturally occurring ethanol-tolerant Lactobacillus species that utilize both glucose and xylose. and L... 2006). the aerobic C. To broaden substrate utilization. and cell mass accumulations (Liu et al. This bacterium can metabolize glucose and xylose simultaneously. 14.3 Bacillus subtilis B. In addition. plantarum (Kleerebezem et al. 2003) will help to predict specific genes to be modified for developing improved biocatalysts for converting biomass into fuel and chemicals.. 2005).

. However. 1988). mobilis ethanol production genes were integrated into the host chromosome via homologous recombination at the ldh locus. This simple temperature change has several industrial advantages including: (i) the reduction of bacterial contamination incidents during ethanol fermentation production campaigns. another engineered strain. naturally occurring thermophilic anaerobic bacteria were studied for ethanol production. with trace amounts of lactic acid (Desai et al.. 2004). 2008). Thermoanaerobacterium saccharolyticum is another thermophilic Gram-positive bacterium that ferments xylan and produces ethanol. Nevertheless... the enzymatic activity levels of these two heterologous enzymes remained too low to permit efficient ethanol fermentation. and Clostridium thermosaccharolyticum (Lynd et al.9 g lÀ1). subtilis BS37.3% (v/v) ethanol after adaptation (Georgieva et al.. as it can ferment at temperatures ranging from 40 to 75  C. in which the L-ldh gene was inactivated. was shown to produce 4–8 g lÀ1 ethanol per hour with 20–40 g lÀ1 glucose/xylose feeds. the Z.4 Thermophilic Anaerobes The advantages of using thermophilic microbes include the possibility to perform simultaneous hydrolysis and fermentation steps at higher temperatures.4. subtilis could potentially be used to reduce ethanol production costs by way of parallel production of valuable products. 1999). Thermoanaerobacter BG1L1. the final ethanol production (4–8 g lÀ1) is comparatively low. lactic acid. Clostridium thermocellum. genetic engineering and gene-transfer systems are required to increase the final ethanol concentrations and eliminate undesired end-products.. However. CO2. ethanologenic B. Recently. thermoglucosidasius strain has potential applications for implementing simultaneous saccharification and fermentation process configurations (Wright et al. in which the Z. strains Clostridium kpu03 and Thermoanaerobacterium kpu04 were reported to produce 1. Notably. (ii) the reduction of ethanol inhibition via product evaporation at higher temperatures. was reported to produce increased acetic acid (1 g lÀ1) and ethanol (1. including Clostridium cellulolyticum. further manipulations of this microbe are needed to increase ethanol production and ethanol tolerance. as most native cellulolytic thermophilic anaerobes carry out mixed-acid fermentations..304 Ethanol and Butanol As a result.0% bean curd refuse when cocultured with a Geobacillus strain (Miyazaki et al. Although heterologous expression of PDC and ADH was achieved in these bacteria. B. subtilis and Bacillus polymyxa to introduce the pet operon (Ingram et al. To accommodate the temperature requirements for hydrolytic cellulases. Bacillus thermoglucosidasius. A genetically modified strain. was reported to produce 9 g lÀ1 ethanol from 20 g lÀ1 glucose (Romero et al. and H2 (Desai et al. which was described as a lactate dehydrogenase-deficient mutant. 2002). Attempts were made to use the Gram-positive hosts B. this resulted in inactivation of the latter gene (Javed et al.. 14. More recently.2 g lÀ1 ethanol from 1. The engineered strain TD1. Electrotransformation protocols have been developed for anaerobic bacteria. 2007).. 2005). mobilis pdc and adh genes were inserted in the chromosome at the ldh (lactate dehydrogenase) site along with inactivation of the als (acetolactate synthase) gene. 2004). acetate. 2007). a thermophilic anaerobe. This patented B. For example. In this strain. this bacterium . and (iii) the reduction of utility usage including energy and cooling water. was reported to tolerate up to 8.

tensions in the petroleum supply chain. microbes other than conventional Saccharomyces have been explored for the efficient conversion of biomass hydrolysates into ethanol and value-added products. Nevertheless. LY168. the clear potential for using immobilized thermophilic anaerobes for continuous ethanol production remains to be tested further at larger scale. varying compositions. Because of the complexities of the lignocellulosic biomass economic value chain (comprising biomass production. Although Saccharomyces has long been used in the starch-based ethanol industry. C25. cerevisiae) fermentation. both of these production objectives. some of which are inhibitors of yeast (S. AX101. the industrial performances of wild-type strains of this microbe remain inefficient for a biomass-based industry. several genetically modified microbes including Z mobilis 8b. mobilis or E. there is a need for the continuous development of engineered bacterial strains that could facilitate the emergence of a biobased industry to achieve. FBR5. and Gram-positive species such as Corynebacterium. Meanwhile. the realization of this vision could very well lead to a sustainable biobased industry in the near future. . in parallel. polyphenolics. Since the simultaneous production of biofuel and coproducts from biomass materials via a biorefinery platform can maximize the economic value of lignocellulosic biomass-based industry. This would in turn benefit not only the environment on a global scale but also the worldwide economy. as it could contribute to significantly reduces ethanol production costs by facilitating simultaneous hydrolysis and fermentation. Already. Nonetheless. biomass hydrolysates usually contain mixed sugars. and their derivatives have shown great promise as ethanologens. Lactobacillus. sugar degradation products.. and recalcitrance to degradation).Conversion of Biomass to Ethanol by Other Organisms 305 exhibits resistance to high acetic acid concentrations (up to 10 g lÀ1) and is capable of fermenting biomass hydrolysates at 70  C. a desired condition to avoid cooling costs and microbial contaminations. This strain utilized undetoxified diluted corn stover hydrolysates and produced 0. Unlike starch. although the fermentation was only carried out in laboratory-scale reactors.. Advances in the field of lignocellulosic biomass conversion will thus continue to include the development of performing bacterial biocatalysts based on the tailor-designed engineering not only of yeasts but also of various bacteria. 2008). Along with cost-effective processes for product separation and recovery. leading to a paradigm shift from the petroleum refinery toward a renewable biorefinery. 2007). including both Gram-negative species such as Z. and other compounds found in plant materials (Bothast et al. Bacillus. and other thermophiles. in combination with environmental concerns. 14. E coli KO11. coli. As a result. or consolidating the bioprocessing into one single reactor (Lynd et al. continue to exert strong pressures.5 Perspective The efficiency of the microbial fermentation of biomass-derived sugar mixtures into fuels and chemicals is one of the chief barriers to the overall economics of cellulosic ethanol industry. the overall biomassto-biofuel commercial production remains a costly process. the development of recombinant thermophilic anaerobes with higher ethanol yields constitute a lead worth exploring further. organic acids. with higher concentrations of biomass hydrolysates.39–0. 1999).42 g ethanol gÀ1 sugars (Georgieva and Ahring.

lactose. Ingram LO (1992) Efficient fermentation of Pinus sp. Cripe J. Potts D. Bothast R. Guerinot ML. Kirk L. Shibata A. Lynd LR (2004) Cloning of L-lactate dehydrogenase and elimination of lactic acid production via gene knockout in Thermoanaerobacterium saccharolyticum JW/SLYS485. Desai SG. acid hydrolysates by an ethanologenic strain of Escherichia coli. Deanda K.0. De Vos WM (1996) Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin. Antoni D. Doelle MB (1993) Zymomonas mobilis – science and industrial application. Doelle MB (1991) Scale-up of ethanol production from sugarcane using Zymomonas mobilis. Conway T (1992) The Entner–Doudoroff pathway in Escherichia coli is induced for oxidative glucose metabolism via pyrroloquinoline quinonedependent glucose dehydrogenase. Fliege R. . Bothast RJ. Ingram LO (1989) Efficient ethanol production from glucose. Bongers R. Biotechnol Prog 15: 867–875. Gobbetti M (2001) The acid-stress response in Lactobacillus sanfranciscensis CB1. J Ind Microbiol 16: 42–47. Crit Rev Biotechnol 13: 57–98. Schwarz WH (2007) Biofuels from microbes. Foster B (2000) Fermentations of pectin-rich biomass with recombinant bacteria to produce fuel ethanol. the USDA neither guarantees nor warrants the standard of the product. Sutton M. Beck MJ. and xylose by recombinant Escherichia coli. Rodriguez Sanoja R. and the use of the names by USDA implies no approval of the product to the exclusion of others that may also be suitable. Appl Biochem Biotechnol 84–86: 181–196. Biotechnol Lett 13: 131–136. Appl Environ Microbiol 71: 1109–1113. Appl Environ Microbiol 55: 1943–1948. Doran JB. Guyot JP (2003) Study of starch fermentation at low pH by Lactobacillus fermentum Ogi E1 reveals uncoupling between growth and alphaamylase production at pH 4. however. Zhang M. Nichols NN. Pallini V. Appl Biochem Biotechnol 84–86: 141–152. Asghari A. Int J Food Microbiol 80: 77–87.306 Ethanol and Butanol Acknowledgments The author thanks Dr Joseph O. Clark DP (1989) The fermentation pathways of Escherichia coli. Loiseau G. Bothast RJ (2000) Development of new ethanologenic Escherichia coli strains for fermentation of lignocellulosic biomass. Crittenden R. Kleerebezem M (2005) High-level acetaldehyde production in Lactococcus lactis by metabolic engineering. Appl Environ Microbiol 58: 1382–1384. Dien BS. Appl Microbiol Biotechnol 65: 600–605. FEMS Microbiol Rev 5: 223–234. Tong S. Ingram LO (1996) Ethanol production from hemicellulose hydrolysates of agricultural residues using genetically engineered Escherichia coli strain KO11. Doelle HW. Dien BS (1999) Fermentations with new recombinant organisms. Names are necessary to report factually on available data. Toh H. Zverlov VV. Bini L. O’Bryan PJ. Fein JE. Doelle HW. Kennedy LD. Nickerson KW. Barbosa MF. De Ruyter PG. and Ms Jacqueline Zane and Ms Karen Hughes for checking the references used in the manuscript. Doran JB. Calderon Santoyo M. Picataggio S (1996) Development of an arabinose-fermenting Zymomonas mobilis strain by metabolic pathway engineering. Appl Environ Microbiol 58: 3826–3829. Flickinger MC (1980) Current biological research on conversion of cellulosic carbohydrates into liquid fuels: how far have we come? Biotechnol Bioeng 22 (Suppl 1): 24–48. References Alterthum F. Microbiology 147: 1863–1873. Rich for reviewing the manuscript and providing valuable suggestions. Nichols NN. Eddy C. Appl Microbiol Biotechnol 77: 23–35. Kuipers OP. Hoefnagel MHN. Appl Environ Microbiol 62: 3662–3667. De Angelis M. Cocconcelli PS. Appl Environ Microbiol 62: 4465–4470.

Park SJ. 0292. Biotechnol Bioeng 83: 854–863. Sandbrink HM. Kerkhoven R. Effects of nutritional supplements. MDF (1999) Ethanol production in gram-positive microbes. Keenan TW. Georgieva TI. Appl Environ Microbiol 53: 2420–2425. Verts AA. Mikkelsen MJ. Verts AA. J Mol Microbiol Biotechnol 8: 243–254. Appl Environ Microbiol 72: 3418– 3428. Shanmugan KT. Siezen RJ (2003) Complete genome sequence of Lactobacillus plantarum WCFS1. Yomano LP. Elsworth Biotech Ltd. Grohmann K. Yukawa H (2004) Metabolic engineering of e Corynebacterium glutamicum for fuel ethanol production under oxygen-deprivation conditions. Conway T (1996) Cloning and expression of the Zymomonas mobilis ‘production of ethanol’ genes in Lactobacillus casei. van Kranenburg R. Cusdin F.Conversion of Biomass to Ethanol by Other Organisms 307 Georgieva TI. Fiers MW. van Sinderen D. Peters SA. Groot MN. Leer R. Jin YS (2003) Metabolic engineering for improved fermentation of pentoses by yeasts. Wyckoff H. Kawaguchi H. Ceigler A (1981) Gamma-ray-induced degradation of lignocellulosic materials. Savoy G. Bron PA. Eggink G. Kuipers OP. Biotechnol and Bioeng XXIII: 2525–2535. Appl Environ Microbiol 62: 4594–4597. Antonie Van Leeuwenhoek 82: 217–235. Inui M. Meagher MM. Tong S. Burgess K. Conway T. Grabar TB. Jarboe LR. Buslig BS (1996) Fermentation of orange peel hydrolysates by ethanologenic Escherichia coli. University of Florida. Cameron RG. Adv Biochem Eng Biotechnol 108: 237–261. Meagher MM. US Patent 5916787. Jeffries TW. Gold RS. Piard JC. Kawaguchi H. Timpa J. de Vos WM. Han YW. Park JP. Smid EJ. Boekhorst J. US patent application 2007. Jensen PR. Groot MN. Ahring BK (2007) High ethanol tolerance of the thermophilic anaerobic ethanol producer Thermoanaerobacter BG1L1. Sybesma W. Appl Biochem Biotechnol 57–58: 383–388. Hutkins RW. Okino S. Kleerebezem M (2002) Metabolic engineering of lactic acid bacteria for the production of nutraceuticals. Inui M. Javed M. Hammer K (1998) The sequences of spacers between the consensus sequences modulates the strength of prokaryotic promoters. Appl Biochem Biotechnol 39-40: 667–685. Kleerebezem M. Yukawa H (2006) Engineering of a xylose e metabolicn pathway in Corynebacterium glutamicum. Ladero V. Gold RS. Appl Environ Microbiol 64: 82–87. Dien BS. Lee SY (2003) In silico prediction and validation of the importance of the Entner–Doudoroff pathway in poly(3-hydroxybutyrate) production by metabolically engineered Escherichia coli. Molenaar D. Ahring BK (2007) Evaluation of continuous ethanol fermentation of dilute-acid corn stover hydrolysate using thermophilic anaerobic bacterium Thermoanaerobacter BG1L1. Lawford HG. Lindsay RC (1967) Dehydrogenase Activity of Lactobacillus Species. Green E (2002) Ethanol production. Preston JF (1987) Genetic engineering of ethanol production in Escherichia coli. Ingram LO. Hols P. Moon SY. Appl Microbiol Biotechnol 63: 495–509. Sesma F. Curr Opin Biotechnol 14: 232–237. Ingram LO. Wisselink W. Lankhorst RM. de Vries M. Sewell GW. Rousseau JD (1993) Production of ethanol from pulp mill hardwood and softwood spent sulfite liquors by genetically engineered Escherichia coli. Barbosa-Alleyne. Conway T (1992) Ethanol tolerance and carbohydrate metabolism in lactobacilli. Ingram LO (2007) Development of ethanologenic bacteria. J Dairy Sci 50: 1585–1588. Tarchini R. Murakami S. Clark DP. Jansen T. Hoffer SM. Hutkins R. Curr Microbiol 33: 256–260..928. Hugenholtz J. Appl Microbiol Biotechnol 77: 61–68. Hong SH. Central Eur J Biol 2: 364–377. Hugenholtz J (2003) Metabolic pathway engineering in lactic acid bacteria. Stiekema W. Proc Natl Acad Sci USA 100: 1990–1995. Kleerebezem M. Ursing B. Hespell RB. Milner P. . J Ind Microbiol 10: 45–54. Bothast RJ (1996) Stabilization of pet operon plasmids and ethanol production in Escherichia coli strains lacking lactate dehydrogenase and pyruvate formate-lyase activities.

Parker C. Wolf Y. Slesarev A. Leathers TD (2008) Lactobacillus buchneri strain NRRL B-30929 converts a concentrated mixture of xylose and glucose into ethanol and other products. Sheehan J. Skinner-Nemec KA. Polouchine N. Smeianov V. Irbis C. Dale BE. Curr Opin Biotechnol 16: 577–583. Israelsen H (1999) Molecular characterization of the pH-inducible and growth phase-dependent promoter P170 of Lactococcus lactis. US Patent 4647534. Lou Y. Pavlov A. Microbiol Mol Biol Rev 66: 506–577. O’Sullivan D. xylose. Barabote R. Himmel M. Wyman CE (2008) How biotech can transform biofuels. Nat Biotechnol 26: 169–172. Weimer PJ. Davison B. Lawford HG. Lynd LR. Lucas S. Lawford HJ (1987) Ethanol production by high performance bacterial fermentation. Nichols NN. Lynd LR. Bransby D. McBride JE. Lawford HG. Cotta MA (2007) Coexpression of pyruvate decarboxylase and alcohol dehydrogenase genes in Lactobacillus brevis. Dowe N. Liu S. Nichols NN. Rousseau JD (2001) Fermentation performance assessment of a genomically integrated xylose-utilizing recombinant of Zymomonas mobilis 39676. Rousseau JD. Cotta MA (2005) Functional expression of bacterial Zymobacter palmae pyruvate decarboxylase gene in Lactococcus lactis. Schell D. Diaz-Muniz I. Zhang M (2002) Cofermentation of glucose. CA). Liu S. Appl Biochem Biotechnol 91-93: 133–146. Welker D. Lee JH. Sorokin A. Dien BS. Cotta MA (2006) Metabolic engineering of a Lactobacillus plantarum double ldh knockout strain for enhanced ethanol production. Appl Biochem Biotechnol 91-93: 117–131. Unlu G. Miyazaki K. Mohagheghi A. Rawsthorne H. Zhang M (2004) Performance of a newly developed integrant of Zymomonas mobilis for ethanol production on corn stover hydrolysate. Lorca G. Mills D (2006) Comparative genomics of the lactic acid bacteria. Ganesan B. Dien BS. Dosti B. Laser MS. (Toronto. Saier M. Goodstein DM. Hawkins T. Koonin E. Curr Microbiol 50: 1–6. Biotechnol Lett 26: 321–325. Pavlova N. Karamychev V. Madsen SM. Rousseau JD (2003) Cellulosic fuel ethanol: alternative fermentation process designs with wild-type and recombinant Zymomonas mobilis. Arnau J. Hamilton R. Lawford HG. Lonvaud-Funel A (1999) Lactic acid bacteria in the quality improvement and depreciation of wine.308 Ethanol and Butanol Lawford HG. Eddy C. Evans K. Hughes J. Liu S. Proc Natl Acad Sci USA 103: 15611–15616. Chou YC. . Hughes SR. George Weston Ltd. Liu S. Tolan JS (2001) Comparative ethanol productivities of different Zymomonas recombinants fermenting oat hull hydrolysate. Lynd LR. Kozyavkin S. Goh Y. Chou YC. Breidt F. Weimer B. Grigoriev I. McMillan JD. Givskov M. and arabinose by genomic DNA-integrated xylose/arabinose fermenting strain of Zymomonas mobilis AX101. Dien BS. Biores Technol 99: 1768–1775. Altermann E. Mohagheghi A. Xie Y. Rousseau JD (2002) Performance testing of Zymomonas mobilis metabolically engineered for cofermentation of glucose. Appl Biochem Biotechnol 105-108: 457–469. Klaenhammer T. J Ind Microbiol Biotechnol 33: 1–7. van Zyl WH. Antonie Van Leeuwenhoek 76: 317–331. Hutkins R. Makarova K. Vrang A. Barrangou R. Mol Microbiol 32: 75–87. Shakhova V. Takada J. Mirkin B. Wechter W. Laser M (2005) Consolidated bioprocessing of cellulosic biomass: an update. Matsuura A (2008) An ability of isolated strains to efficiently cooperate in ethanolic fermentation of agricultural plant refuse under initially aerobic thermophilic conditions: oxygen deletion process appended to consolidated bioprocessing (CBP). FEMS Microbiol Lett 274: 291–297. van Zyl WH. Bischoff KM. Keller M. Pretorius IS (2002) Microbial cellulose utilization: fundamentals and biotechnology. Steele J. xylose. Plengvidhya V. Rohksar D. Appl Biochem Biotechnol 98–100: 885–898. Tamir D. Broadbent J. Richardson P. and arabinose. Huang K. Appl Biochem Biotechnol 98-100: 429–448. J Ind Microbiol Biotechnol 35: 75–81. Benson A. Baldwin K.

Lee HJ. Int J Food Microbiol 89: 105–124. van Maris AJ. Bengtsson O. Zacchi G (2006) Simultaneous saccharification and co-fermentation of glucose and xylose in steam-pretreated corn stover at high fiber content with Saccharomyces cerevisiae TMB3400. Kang HL. Kang HS (2005) The genome sequence of the ethanologenic bacterium Zymomonas mobilis ZM4. Jeon YJ. Romero S. Nichols NN. Ingram LO. Gosset G. Sun Y. Lee KJ. Jin SJ. Gil MA. J Ind Microbiol Biotechnol 30: 315–321. Okuda N. Yomano LP. Talarico LA.Conversion of Biomass to Ethanol by Other Organisms 309 Mohagheghi A. Takao M. Winkler AA. Process Biochem 41: 1806–1811. J Ind Microbiol Biotechnol 30: 279–291. Appl Environ Microbiol 73: 5190–5198. Verts AA. van Dijken JP. Lee JS. J Biosci Bioeng 103: 350–357. Arch Microbiol 179: 227–233. Ingram LO. Hong JH. Biotechnol Lett 1: 165–170. Talarico LA. Neidhardt FC. Galbe M. Ohgren K. Adv Biochem Eng Biotechnol 108: 263–288. Park HS. Yoon KO. Oh SJ. Unden G (2005) Pyruvate fermentation by Oenococcus oeni and Leuconostoc mesenteroides and role of pyruvate dehydrogenase in anaerobic fermentation. De Ley J (1977) The biology of Zymomonas. Nat Biotechnol 23: 63–68. Konings WN (2003) Beer spoilage bacteria and hop resistance. Um HW. Sunderland. Merino E.292. Adv Biochem Eng Biotechnol 108: 179–204. Beall DS. Microbiology 151: 4023–4031. Chong H. Ingraham JL. . Seo JS. Richter H. Lawford HG (2007) Zymomonas mobilis for fuel ethanol and higher value products. Park CJ. de Laat WT. Hamann I. Kuyper M. Kil JI. J Mol e Microbiol Biotechnol 15: 16–30. Yukawa H (2008) Technological options for biological fuel ethanol. Bothast RJ (2003) Engineering lactic acid bacteria with pyruvate decarboxylase and alcohol dehydrogenase genes for ethanol production from Zymomonas mobilis. Kim JH. Kim JY. Sakamoto K. Lee KJ. Hahn-Hagerdal B. Maupin-Furlow JA (2001) Production of the Gram-positive Sarcina ventriculi pyruvate decarboxylase in Escherichia coli. Selzer PM. Rogers PL. Mejia JP. Schaechter M (1990) Physiology of the Bacterial Cell: A Molecular Approach. Rutherford WM. Rogers PL. Appl Environ Microbiol 71: 4966–4971. US Patent Application 2006. Ruth M. MA. Biores Technol 83: 1–11. J Biotechnol 126: 488–498. Cheng J (2002) Hydrolysis of lignocellulosic materials for ethanol production: a review. Saha BC (2003) Hemicellulose bioconversion. Tran QH. Richter H. Martinez A (2007) Metabolic engineering of Bacillus subtilis for ethanol production: lactate dehydrogenase plays a key role in fermentative metabolism. J Bacteriol 57: 147–158. Bacteriol Rev 41: 1–46. Appl Environ Microbiol 57: 893–900. Shioya S (2007) Microaeration enhances productivity of bioethanol from hydrolysate of waste house wood using ethanologenic Escherichia coli KO11. Inc. Unden G (2003) Use of the mannitol pathway in fructose fermentation of Oenococcus oeni due to limiting redox regeneration capacity of the ethanol pathway. Gorwa-Grauslund MF. Pronk JT (2007) Development of efficient xylose fermentation in Saccharomyces cerevisiae: xylose isomerase as a key component. Microbiology 147: 2425–2435. Sinauer Associates. Spielbauer AJ (2006) Ferrulate esterase producing strains and methods of using same. Kim H. Tribe DE (1979) Kinetics of alcohol production by Zymomonas mobilis at high sugar concentrations. Ninomiya K. Wagner N. Swings J. Inui M. Katakura Y. Shanmugam KT. Maupin-Furlow JA (2005) Construction and expression of an ethanol production operon in Gram-positive bacteria. Stokes JL (1949) Fermentation of glucose by suspensions of Escherichia Coli. Dien BS. Schell DJ (2006) Conditioning hemicellulose hydrolysates for fermentation: Effects of overliming pH on sugar and ethanol yields. Ingram LO (1991) Genetic improvement of Escherichia coli for ethanol production: chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II. Smiley BK. Bolivar F. Kim JJ. Ohta K. Lee KJ. Lee SY. Jung C. 0046. Oh HM. Nsereko VL.

Cotta MA (1997) Cloning. Finkelstein M. Finkelstein M (1998) Single Zymomonas mobilis strain for xylose and arabinose fermentation. 61–67. DC pp. York SW. Chow J. Wyman CE. Shanmugam KT. Solar Energy Research Institute. McMillan J. Deanda K. Franden MA. Finkelstein M.310 Ethanol and Butanol Wang DIC. Ingram LO (1998) Isolation and characterization of ethanol-tolerant mutants of Escherichia coli KO11 for fuel ethanol production. Wright JD. Zhang M. Picataggio S (1995) Promising ethanologens for xylose fermentation. sequence. Whitehead TR. J Ind Microbiol Biotechnol 20: 132–138. Washington. Wyckoff HA. Biocic I. Appl Biochem Biotechnol 51/52: 527–536. Biotechnol Lett 27: 1891–1896. In Proceedings of the Third Annual Biomass Energy Systems Conference. Fang HY. Curr Microbiol 34: 367–373. Department of Energy. Picataggio S (1995) Metabolic engineering of a pentose metabolism pathway in ethanologenic Zymomonas mobilis. and expression of the L-( þ ) lactate dehydrogenase of Streptococcus bovis. Zhang M. Midwest Research Institute. Yomano LP. Zhou S. Kansas City. Eddy C. Wang SD (1977) Direct microbial conversion of cellulosic biomass to ethanol. Zhang M. . Chou YC. United States Patent 5843760. Newman M. Picataggio S. Grohmann K (1988) Simultaneous saccharification and fermentation of lignocellulose: process evaluation. MO. Appl Biochem and Biotechnol 18: 75–89. Ingram LO (2005) Fermentation of 10 % (w/v) sugar to D-(À)-lactate by engineered Escherichia coli B. Yomano LP. Science 267: 240–243.

Nonetheless.1 Introduction Today’s industrial fermentation processes are largely classified as batch. all of which are used to drive the conversion of sugars and starch-based materials into valuable products. and immobilized cell systems. however. Although each of these processes has become well established. The decision as to which fermentation processes should be implemented in a particular plant involves weighing the advantages and disadvantages of each of these four classes of process. it is primarily the fed-batch (and to a lesser extent continuous) mode of operation that is dominant in today’s ethanol industry. the necessary investment and operating costs. several processes that integrate two or more aspects of these various ethanol production processes are described. Verts and Hideaki Yukawa e 15. Finally. fed-batch. in conjunction with cell recycling membrane systems [1]. much emphasis is placed worldwide on achieving a critical manufacturing capacity for bioethanol as a strategy to reduce CO2 emissions – and therefore to combat Biomass to Biofuels: Strategies for Global Industries and Hideaki Yukawa Ó 2010 John Wiley & Sons. including a higher productivity [2] and an improved tolerance to fermentation inhibitors [3]. Alain A. growth-arrested processes present several intrinsic advantages. the major guiding principle for practitioners in the field remains the implementation of a process that requires the minimum capital costs per unit of product recovered. as well as the yield and productivity levels desired. This includes examining the properties of the primary raw materials used. Hans Blaschek e . ethanol production processes may employ growth-arrested cells at very high cell densities. Currently. Ltd Edited by Alain Verts. In addition. When compared to existing fermentation processes.15 Advanced Fermentation Technologies Masayuki Inui. the technological complexity of the processes and availability of a competent workforce. continuous operation. Nasib Qureshi.

the substrate and cells (which are . simultaneous saccharification and fermentation (SSF). whilst CBP undoubtedly represents the biggest technological challenge. In a further twist. as this would not only help to reduce the pressure on food crops but also permit a noncontroversial increase in the global demand for bioethanol [4. 7]. an industrial challenge has arisen to use selective hexose-fermenting organisms such as Saccharomyces cerevisiae to ferment lignocellulosic hydrolyzates. and are usually slower than hexose fermentations. In contrast. Although most processes selectively hydrolyze C6 sugars. only when the hexose concentrations have fallen appreciably will the pentoses begin to be consumed. However. cause the process to become much more complex [10. Thus. 7]. as the chief ingredients of holocellulose. Processes such as separate enzymatic hydrolysis and fermentation (SHF). such that in order for the fermentation process to be economically successful it must not only be optimally designed but also well controlled within a relatively narrow range of operational parameters. it also represents the largest potential economic gains by closely integrating many of the different steps of ethanol production. all bear the potential for dramatic economic gains by combining enzymatic hydrolysis and fermentation in a single step. Lignocellulosic hydrolyzates contain a mixture of pentose (C5) and hexose (C6) sugars. Unfortunately.312 Ethanol and Butanol global warming – while simultaneously reducing our reliance on fossil fuels for transportation [4. the fact that feedstocks for bioethanol production currently derive from the edible parts of food crops. the fermentation of hexoses by these organisms typically occurs at a high ethanol yield and productivity. For this. 6. and only under anaerobic or microaerobic conditions. and without any lag phase. in order to maximize the overall process yields and to avoid problems in wastewater treatment. pentose fermentations generally progress under aerobic conditions. the ‘pretreatment technologies’ required to delignify this fibrous material lead to the creation in the resultant lignocellulosic hydrolyzates of fermentation inhibitors that. 15. such as lignocellulosic biomass. generally by causing the residence times to be longer than they would had all the sugars been used simultaneously. has led to an undesirable competition between bioethanol production and food supply [6. Yet. This so-called ‘carbon catabolite repression’ (CCR) phenomenon may have a negative impact upon productivity. as land for the cultivation of biofuel crops is increasingly carved out of the tropical rain forests [8. The use of lignocellulosic biomass as raw materials. 9].2 Batch Processes To the present day. If microorganisms are capable of fermenting both types of sugars. it is important that pentoses such as xylose and arabinose should also be utilized. engenders a variety of technical barriers that must first be overcome. such as sugar cane or maize. many of them will utilize the hexoses first. in turn. This latter phenomenon further complicates the entire process. 11]. and are maintained in a medium containing a mixture of hexoses and pentoses. however. Indeed. most biotechnological ethanol is produced essentially by the same processes that have been used for centuries by the beverage industry and involve a simple batch fermentation of carbohydrate feedstocks. 5]. as well as the concept of consolidated bioprocessing (CBP). is clearly desirable. A switch to more abundant primary raw materials. this situation is now variously blamed not only for creating dramatic increases in food prices but also for exacerbating global warming-inducing deforestation.

if unusually high amounts of inhibitors are present in the hydrolyzate they may not be detoxified and cell growth may be totally prevented. techniques for cell reuse and the use of series of several fermentors have been implemented. the drawback of this simple design is the significant idle time that is required while the bioreactor is emptied. the conversion efficiency attained by yeasts lies in the range of 90–95% of the theoretical value. If the lignocellulosic hydrolyzate used is well-detoxified. at which point the fermented material is pumped to a distillation column. However. (ii) easy operations. the cells may be filtered and collected from the filter by using hydrocyclones. Nevertheless. as used in most Brazilian distilleries. (iv) the use of less-qualified labor. the concentrations of the cells. In order to improve on conventional batch processes. Among the advantages inherent to the simplicity of the batch process are: (i) low investment costs. the economic advantage of using a single reactor to generate various products may be offset by the increased risk of infection or mutation of microorganisms. if necessary. on the other hand. then near-optimum growth characteristic and superior productivity can be expected. On completion of the fermentation phase. or does not contain any fermentation inhibitors. nutrients. Nonetheless.. even in such cases. Notably. or when the commercial value of the products thus manufactured is high. If.g. substrates (e. Most importantly. As an alternative to centrifugation. if operated at staggered intervals. while the overall process is of limited productivity due to the relatively long downtimes between batches and the existence of a lag phase at the start of each new fermentation batch. One clear drawback of this latter plant design is the need for a higher initial capital expense. During cultivation. Depending on the characteristics of the carbohydrate feedstock. sterilized. and (vii) reduced risks of infection and cell mutation due to the relatively short duration of each cultivation period. vitamins) and products will be changed. cooled. can provide a continuous feed to the distillation system. Chapter 12). (iii) a low requirement for complete sterilization. cleaned. several . during which a proportion of the inhibitors will be detoxified (metabolized). and a new batch is introduced. sterilized. Such alterations slow rapidly towards the end of the cultivation period. with a final ethanol concentration of 10–16% (w/v) [4]. the bioreactor is washed.Advanced Fermentation Technologies 313 grown separately) are introduced into a bioreactor together with nutrients and. the recycling of cells can increase the overall process productivity. Yeast cells from previous fermentations are separated from the media by centrifugation. the hydrolyzate contains even small amounts of inhibitors. (v) lower risks of financial loss. the batch process is the most preferred when only small amounts of products are required. this method allows up to 95% of the yeast cells to be recycled [13]. then a relatively long lag phase may ensue. additional enzymes. Despite these disadvantages. This process achieves a reduced fermentation time and an increased yield by recycling the yeast while simultaneously employing several fermentors operated at staggered intervals. frequent sterilizations are necessary. one of the most successful batch systems applied to the industrial production of ethanol is the ‘Melle-Boinot’ process. However. it is vital to consider the presence of any fermentation inhibitors that might hinder efficient ethanol production. and/or the product contamination that may occur during different production campaigns. and up to 80% recycled [12]. carbon source. Although a variety of treatments have been investigated to mitigate the presence of such fermentation inhibitors in the media during ethanol production (cf. When a lignocellulosic biomass is used as the raw material. while the use of several fermentors in series. (vi) easy management of feedstocks. heated and recharged.

the use of mutants that are tolerant against the inhibitors (through either adaptation or genetic engineering modifications). This is achieved by intermittent feeding of the substrate. These processes are characterized by high-yields in standard manufacturing environments. and optimized fed-batch processes. fed-batch processes with feedback control are subdivided into indirectcontrol and direct-control fed-batch processes [14]. with a substrate and all other required nutrients being added continuously or intermittently to the initial medium. fed-batch processes have largely solved the problems of substrate inhibition or catabolite repression by maintaining low substrate concentrations in the bioreactor. On the other hand. and the relatively long downtime required for filling. given their high flexibility and well-defined cultivation times (as no cells are added or removed during the cultivation period). or at a point halfway through the batch process. As noted above. constant-rate fed-batch. is one of the most popular processes in the bioethanol industry. The substrate concentration is maintained constant in the reactor by substrate feeding. Consequently. Fed-batch processes without feedback control can be classified further into intermittent fed-batch.314 Ethanol and Butanol strategies might also be considered to improve detoxification in situ and obtain a higher ethanol yield and productivity. which can be regarded generically as a combination of batch and continuous operations. In this way. Adding the substrate at the same rate at which it is consumed allows substrate inhibition to be minimized. the use of such processes enables the problems of mutation and plasmid instability. a highly qualified workforce is required to successfully conduct fed-batch manufacturing operations. and leads to increased productivities. Even with these disadvantages. ethanol) is attained. the downside of a fed-batch design is that it requires the use of complex (and expensive) control systems. heating. Conversely. and the corresponding feed flows adjusted accordingly. In addition. which are frequently encountered in continuous cultures. and also to handle any process deviations that inevitably occur in the absence of feedback control. The start-up of a fed-batch operation is similar to that of a batch operation.g. These include the use of cells at high initial cell densities. fed-batch processes are often applied when continuous processes are impractical. exponential fed-batch. fed-batch processes do not suffer from any of the cell washout problems that typically occur in continuous fermentations. The substrate and nutrient concentrations can be measured by using online probes. while the effluent is removed at relatively regular intervals. for example when there is a significant risk of the . although the concentrations of the nutrients and substrates are maintained at low levels. 15. they are sufficiently high for optimum productivity. to be avoided. emptying and cleaning the reactor.. sterilizing. The process is continued until a different nutrient becomes limiting and/or an inhibitory concentration of the product (e. This may be carried out either at the start of the cultivation period.3 Fed-Batch Processes The fed-batch process. for example when the time courses do not follow expected profiles. Furthermore. as well as the excessive wear and tear on instruments due to frequent sterilization. cooling. and/or the development of optimal reactor conditions so as to minimize the effects of the inhibitors.

the feed rate can be increased when starting from a higher initial cell concentration. Notably. As long as any substrate addition occurs at a low rate in a fed-batch process. 19]. . the feed medium containing all the nutrients is fed continuously at a constant rate. during the process. the culture medium and other required nutrients are pumped continuously (with agitation) into a bioreactor where the microorganisms are already active.. Hence. the success of a fermentation batch will depend heavily on the feed rate of the hydrolyzate. large amounts of sugars are consumed. In continuous fermentation without feedback control (also known as a ‘chemostat’). while the cultured broth is simultaneously removed from the fermenter at the same rate. A turbidostat with feedback control is a continuous process where the cell concentration is maintained at a constant level by controlling the medium feeding rate. ethanol). Continuous fermentations with feedback control include turbidostats. The fermentation mixture containing the product (e. With regards to the fermentation of hydrolyzates from lignocellulosic materials. The major advantage of this technique is the ability to conduct an in situ detoxification of the hydrolyzates by a direct action of the fermenting microorganisms. The chemostat format is quite useful for optimizing media formulations and/or for investigating the physiological state of microorganisms [14]. In the bioreactor. phauxostats. a too-low feed rate might result in the hydrolyzate being converted. Continuous fermentation operations may be further classified as with or without feedback control. the applicability of fed-batch processes has been well established [15–18]. and thus to maintain a high cell growth rate. 15. or when using organisms that exhibit a greater tolerance against common inhibitors. the feeds containing the substrate. The process is operated in such a way that the composition of the solution in the bioreactor remains constant. as the use of an inhibiting hydrolyzate at a very high feed rate would limit both ethanol production and cell growth. cells and residual sugars is continuously collected from the top of the bioreactor. the importance of operating at an optimum feed rate cannot be overemphasized [15–17. hydroxymethyl furfural) will be kept low in the bioreactor. limiting their inhibitory effects. new cells are continuously grown while older cells are continuously washed out. furfural.4 Continuous Processes Continuous ethanol production considerably reduces industrial inputs such as operator time or equipment downtime. However. In this way. In continuous processes.. Air is supplied continuously to the bioreactor in order to ensure a high level of oxygenation. because yeast has only a limited ability for converting inhibitors.g.g. the concentrations of bioconvertible inhibitors (e. in contrast. and large amounts of ethanol and new cell mass are produced. such that the effects of most inhibitors would be minimized. or when a batch processes would result in too low a productivity. and nutristats [14]: . Another interesting pathway for optimization would be to use optimal reactor conditions. and a steady state is achieved when the growth and washout rates are equal. the substrate concentration appears to be a critical parameter. but at a very low productivity [17]. Hence.Advanced Fermentation Technologies 315 microorganisms becoming mutated or infected.

For example. The second fermentor. as it is extremely difficult to adapt the operating conditions of such systems to large process fluctuations. so as to avoid cell washout. cell growth would need to be maintained at a rate equal to the dilution rate. the microorganisms used for the fermentation of lignocellulosic hydrolyzates normally lose their activity after prolonged exposure to the inhibitory conditions of the hydrolyzate. a characteristic which is extremely helpful for large-scale productions. Despite these major advantages of continuous systems. as the operating conditions are fixed. the product quality is more consistent and less damage is caused to the instruments by sterilization. it is especially crucial that the cells maintain their viability and activity over a long period of time. medium composition. can convert less sugar than if operated . any ethanol-mediated inhibition would be reduced in the first unit. Ethanol can be produced by using continuous-flow fermentor units arranged in series. and employing only those microorganisms that have a high stability against mutation. In order to tailor continuous processes for fermenting lignocellulosic hydrolyzates. despite one of the major advantages of continuous cultivation being the possibility of running the process for long periods of time. Taken together. The major drawback of lignocellulosic hydrolyzates in continuous fermentation is that. Moreover. In contrast. leads to high investment costs. Although continuous fermentation systems aim at maintaining high fermentation rates. despite the potential productivity advantages offered by this technology. the low growth rate typically exhibited by cells during the fermentation of lignocellulosic hydrolyzates would need to be accounted for. temperature or oxygen concentration are possible. the retention time can be chosen so that fermentation of the sugar occurs partially in the first unit. where the pH value of the medium in the fermenter is maintained at a preset value. they demonstrate stringent requirements with regards to the raw material quality. using expensive control and automation equipment. it is particularly important that the raw material quality is kept relatively constant. When two fermentors are arranged in series. continuous fermentation techniques have until now not been widely adapted to the use of lignocellulosic hydrolyzates. Given that lignocellulosic hydrolyzates all contain certain levels of fermentation inhibitors that may arrest the fermentation process. at a very low dilution rate. In this way.316 . allowing a faster throughput. In fermentations that occur in the presence of fermentation inhibitors. There is also a high risk that the microorganisms used may undergo mutational changes over long cultivation periods. Ethanol and Butanol A phauxostat is an extended nutristat. Continuous operations are easier to mechanize and automate than batch or fed-batch processes. these conditions have led to continuous fermentation processes being preferred only for products that both demand high production rates for economic viability. A nutristat with feedback control is a cultivation technique where the nutrient concentration is maintained at a constant level. providing complete sugar utilization and high ethanol concentrations. Moreover. they exhibit a low degree of flexibility as only small changes in throughput. As a result. continuous processes require less manpower and smaller reactor volumes as the downtime periods are shorter. . the conversion rate of the inhibitors would be expected to decrease due to the reduced specific growth rate of the biomass. with its lower productivity. washout may occur even at very low dilution rates. consequently. and is completed in the second unit. Moreover. the need for continuous medium sterilization.

at high product concentrations a two-stage system may be up to 2. and (iii) expensive processes of cell recovery and cell recycling are not required. This is a simple. Cell immobilization processes are not always optimal. 22].. In turn. Another problem with porous support materials is in the control of microenvironmental conditions. to attain the maximum rate of bioconversion.5. Notably. these local conditions result in an enhanced overall performance of the cell biocatalysts (e. Therefore. 15. biomass growth and gas evolution may cause severe problems in some systems. thereby restricting the rate of product secretion or the rate of substrate uptake by the cells. high volumetric productivities can be achieved given the high cell concentrations and high flow rates. Consequently. In addition. Nevertheless. Although high cell loadings can also be achieved using microporous support materials.1 Surface Attachment In immobilization by surface attachment. based on the physical mechanism of cell localization and the nature of support mechanisms: (i) attachment to a surface. (iii) containment behind a barrier. the cells are allowed to attach to a solid support. an optimal pore size should be selected. Immobilization can be defined as the restriction of cell mobility within a defined space. . As a result. though this has been shown to be improved [21. the accessibility of the nutrient to the inner surfaces of pores may be the limiting factor.5 Immobilized Cell Systems Ethanol production from biomass by fermentation is possible using either free or immobilized cells. but limited cell loadings and weaker binding forces reduce the utility value of this method. nutrient–product gradients. however. The major benefit of cell immobilization by surface attachment is the provision of direct contact between the nutrient and support materials. their performances may be limited by low rates of exchange between the immobilization matrix and the fermentation medium.3-fold more productive than its single-stage counterpart [20]. and (iv) self-aggregation [23]. 15. immobilized systems minimize shear damage and permit the development of favorable microenvironmental conditions such as cell–cell contact. including significant mechanical disruption of the immobilization matrix. or pH gradients. four types of immobilization technique have been recognized. using a wide variety of different carrier materials [24]. while the specific surface area may be the limiting factor at large pore sizes. (ii) they are not susceptible to cell washout problems at high dilution rates.Advanced Fermentation Technologies 317 alone. higher product yields and rates). the key to a successful immobilized cell process is the genetic stability of the immobilized cells. In the case of small pore sizes. this may lead to intraparticle pore diffusion limitations at high cell densities. Optimum commercial fermentation designs must retain cell viability while repressing excessive vegetative growth of the producing microorganism and the formation of undesired products that may accompany growth. In general. (ii) entrapment within a porous matrix. Immobilized cell cultures have the following advantages over suspension cultures: (i) they permit higher cell concentrations. as seen with polymer-entrapped cell systems [24].g. but will convert most (if not all) of the sugar present. Moreover. low-cost method of cell immobilization.

which is synthesized in situ around the cells [29]. should be sufficiently porous so as to allow the transport of substrates and products in and out of the bead.31