For most of the two decades after 1980, the scientific debate on ethanol and other biofuels revolved around the issue of production cost relative to those of conven­tional gasoline and diesel. As late as 1999, a lead author of the NRC report was still focusing on the question: is there any real hope that biobased products can compete economically with petroleum-derived products?108

Less than a decade on, a radically different perspective on biofuels has been enforced by the dramatic increase in oil prices that have rendered quite irrelevant the doubts expressed in the 1980s and 1990s on the feasibility of ever producing biofuels at an economic cost competitive to that of conventional gasoline. This watershed was evident in a comment made in an article in the August 29, 2006, issue of the International Herald Tribune: “As long as crude oil is above $50 a barrel, there is a momentum to biofuels that is unstoppable.”109 Analyses made in the 1980s and 1990s (and earlier) were all individually correct in that they reflected the prevailing economic realities of world oil prices; whatever the production route, bioethanol and other biofuels were only likely to be palatable as mass transportation fuels if heavily subsidized and/or as a result of enthusiastic, deliberate, and sustained governmental action (as in Brazil). In late 2007, the future of biofuels seems even rosier as world oil prices have topped first $80 a barrel, then $90 a barrel, and are predicted by industry analysts (never adverse to risking the “ridicule and resentment” noted at the begin­ning of this chapter) to even reach $120 a barrel during 2008.

Conversely, it is straightforward to identify the conditions under which the per­ceived “momentum” in favor of biofuels would falter:

• A severe (or sustained) global economic recession, or

• An unexpected announcement of the discovery of several major untapped oil deposits in politically stable regions of the world and that could be exploited at costs no more than marginally above those presently accepted by the oil industry

Both of these events would act to reduce crude oil prices again toward (if not to) those “enjoyed” for most of the twentieth century (figure 1.3). The outcome would be, however, no more than a postponement of an “event horizon” of far greater historical significance than the year-on-year fluctuations of oil and gasoline retail prices.

The concept of ultimate cumulative production, now usually referred to as estimated ultimate recovery, of global oil was introduced in 1956.110 Given a finite quantity of oil in the Earth’s crust, production could only reach that fixed amount, the oil extraction rate being mathematically fitted by a curve function with a dis­tinct maximum, that is, the “peak oil” theory. By 1956, historical oil production rate maxima were well known: for the Ohio oil field before 1900 and for the Illinois oil field in 1940; projecting forward, the U. S. peak production rate was predicted to occur between 1970 and 1975 and a world peak production rate around the year 2000 — in contrast, world coal production would have a much delayed peak rate (approximately 2150), and total fossil fuel “family” (oil, coal, and natural gas) might last until 2400-2500.[56]

Although U. S. oil production did, in the event, peak a little before 1975, the global picture has remained unclear, and “oil prophets” (who are equally as capable of producing reactions of deep skepticism and dread as are economists) now predict, in reports and publications appearing between 1997 and 2007, peak oil rates to occur at any date between 2010 and 2120, with a mean value of 2040.111 The great variabil­ity in these rival estimates derives from multiple uncertainties, including those of the extent of future discoverable oil reserves and their timing. The onset of irreversible decline could also be influenced by developments in oil-producing regions, in par­ticular increasing domestic oil consumption might eliminate over time the ability of some countries to export oil to net consumers, reducing the number of net exporters from 35 to between 12 and 28 (another large uncertainty) by 2030 — there is even the “inverse” oil security scenario where Middle Eastern oil exporters attempt to withhold or restrict oil extraction if faced by a concerted attempt on the part of the OECD to reduce dependence on Middle Eastern oil.112

To return to a question posed in the preface, when will the oil run out? A dif­ferent perspective is provided by calculating the quotient of the known oil reserves and the actual consumption rate, accepting that “proven” oil reserves may be over­estimated or underestimated and putting to one side any possible (but unproven) reserves to be substantiated in the future (figure 5.13). For the past 18 years, this estimate has changed little, after increasing markedly between 1979 and 1998 as new discoveries were made; the average time until exhaustion of the supply has been

41.5 years (with a standard deviation of ±1.0 year) since 1998. The dilemma lies in interpreting the detailed trend line: is the mean “life expectancy” of oil reserves now decreasing? After reaching parity in the late 1980s, the rate of discovery has been overtaken by the consumption rate since 2003; if that relative imbalance per­sists, the original Hubbert prediction will prove to have been accurate (figure 5.14). There certainly is no sign of the time to eventual exhaustion having increased during the past 20 years, individual years of optimism being followed by a succession of years that fit better with a static or decreasing trend (figure 5.13). The Energy Watch Group, a German organization “of independent scientists and experts who investi­gate sustainable concepts for global energy supply,” concluded in October 2007 that

so many major production fields were past their peak output that oil supply would decrease rapidly from 81 million barrels/day in 2006 to 58 million barrels/day by 2020 to 39 million barrels/day by 2030, with all regions (apart from Africa) showing reduced production rates by 2020, that is, “peak oil is now.”113 Table 5.21 collects the Energy Watch Group’s analysis of the historical sequence of individual nations’ peaks of production since 1955 and compares these dates with the dwindling rate of new oil field discoveries after the 1960s. As collateral, the evidence was presented in the German report that big international oil companies, taken in aggregate, have been unable to increase their production in the last decade despite the marked rise in world oil prices. Coincidentally, in October 2007, oil prices exceeded $90/barrel, highlighting the persistence of the new era of high energy prices that bodes ill if diminishing oil supplies seriously exert their inevitable economic effect.

With natural gas supplies, the horizon of exhaustion remains further away, that is, with a mean value of more than 66 years for estimates made after 1988 (figure 5.15). Integrated over the past 19 years, however, the outlook does not offer promise of natural gas supplies having an increased longevity. If anything, the prospect appears

to be one now of rapidly dwindling stocks if the trends from 2001 onward prove to be consistent (figure 5.15). As with oil, therefore, the era of discovery of large and accessible reserves may be over.

Unless fuel economy is radically boosted by technological changes and popular take-up of those choices, price pressures on oil products caused by a dwindling or static supply (and an expected increase in demand from expanding Asian economies) will act to maintain high oil and gasoline prices. Modeled scenarios envisaged by the DOE and European energy forums include those with persisting high oil prices[57]

can bear, and calling upon a wider range of socioeconomic factors to develop a full accounting of “externality costs.”115 A clearly positive case can, however, now be made without the recourse to such arguments.

Although there are nonbiological routes to substitutes for petroleum products, capable of extending liquid hydrocarbon fuel usage by a factor of up to tenfold, their estimated production costs lie between two — and sevenfold that of conven­tional gasoline and oil products (figure 5.16).116 Unavoidably, their production from oil shale, tar sands, natural gas, and coal deposits would add massively to total greenhouse gas emissions. Although it is also possible that technological innova­tions will enable oil to be extracted with high efficiency from such nonconventional sources as oil shale and tar sands and push the limits of geographical and geologi­cal possibilities for neglected or undiscovered deep-ocean oil deposits, these too will be costly.[58]

Perhaps, the crucial debate in coming decades will be that of allocating public funds in the forms of tax incentives to offset exploration costs (on the one hand, to the oil industry) or R&D costs to a maturing biofuels industry, that is, the crucial policy decisions for investments in new technologies that may be substan­tially unproven when those choices must be made. The oil industry has, in fact, been highly successful in attracting such subsidies. The last (numerical) word can be left with the U. S. General Accountability Office, whose assistant director for Energy Issues, Natural Resources, and the Environment made a presentation to the “Biomass to Chemicals and Fuels: Science, Technology, and Public Policy” conference at Rice University, Houston, Texas, in September 2006.117 Tax incen­tives to the ethanol industry between 1981 and 2005 amounted to only 12% of those for the oil and gas industry between 1968 and 2005 (figure 5.17). These figures underestimate the full sums expended in incentives to the oil and gas industry because they date only from when full records were kept by the U. S. Treasury of revenue losses, not when an incentive was implemented (the Tariff Act of 1913, in the case of the oil and gas industry). Although the magnitudes of some subsidies for conventional fuels are much reduced presently as compared with the situation in the 1970s and 1980s, they still outweigh the sums laid out to support biofuels. An “incentives culture” has, therefore, a long history in shaping and managing energy provision.

The competition is not between fuel ethanol, on the one hand, and substitutes for conventional oil products, on the other, but that between rival technologies for liquid fuels (most of which are biobased) and — a highly strategic issue — using plant biomass as either a source of biofuels or predominantly a source of carbon to replace petrochemical feedstocks in the later twenty-first century. Is the future one of a hydrogen economy for transportation and a cellulose-based supply of

“green” chemicals? Will nuclear power be the key to providing hydrogen as an energy carrier and will biological processes provide liquid fuels as minor, niche market energy carriers for automobiles? Or is the future bioeconomy (as discussed in chapter 6) a mosaic of different technologies competing to augment nuclear, solar, and other renewable energy sources while gradually replacing dwindling and ever more expensive hydrocarbon deposits as a renewable and (possibly) sus­tainable bedrock for the chemical industry beyond the twenty-first century?

The conference, entitled Cellulosic Ethanol and 2nd Generation Biofuels, was held over three days in October 2007 and focused on scientific and economic issues beyond those of starch — or sucrose-derived fuel ethanol. Itemized separately, the presentations and panel discussions were [68]

• Development of cellulosic ethanol — perspective from the European Union

— see chapter 5, section 5.2.7

• Commercial development of cellulosic ethanol — steps toward industrial — scale production — see chapters 4 and 5

• Tapping the potential of forest products as a feedstock for cellulosic ethanol

— see chapter 4, section 4.2.6, and chapter 5, section 5.4

• Logistics of switchgrass as a cellulosic feedstock — see chapter 4, section 4.2.2, and chapter 5, section 5.4

• Overcoming resource and transportation constraints for cellulosic ethanol

— see chapter 4, section 4.2, and chapter 5, section 5.4

• Feedstock procurement and logistics for cellulosic ethanol — see chapter 4, section 4.2, and chapter 5, section 5.4

• Advances in pretreatment and cellulosic ethanol production — see chapters 2 and 4

• Enzymes and ethanologen challenges for cellulosic ethanol — see chapter 2, sections 2.5 and 2.6, and chapter 3

• A different approach to developing yeasts for biomass conversion — see chapter 3, sections 3.1, 3.2, and 3.4

• Siting and financing a cellulosic ethanol plant — see chapter 5

• Dealing with legal and policy risks for ethanol and advanced biofuels — see chapter 5, especially section 5.2.2

• Investor perspectives on developing and financing cellulosic ethanol — see chapter 5

From this agenda, it is evident that, unlike starch — and sucrose-derived ethanol indus­tries (very much still in the “boom” phase of development worldwide), cellulosic eth­anol still remains on the fringes even in the developed economy (the United States) with the most overt public support of cellulosic ethanol while investors fret about fiscal uncertainties and technologists scrabble to convince potential manufacturers that the major scientific hurdles have been overcome. In New Zealand, the Genesis Research and Development sold (in early November 2007) BioJoule, its biofuels business that aimed at establishing a lignocellulosic ethanol and biorefinery facility, citing the difficulties in raising capital and grant funding to pursue such an aim.

Other than cellulosic ethanol, other advanced biofuels (with the exception of “biobutanol”) remain in need of serious industrial partners or timelines. The obvi­ous danger to many environmentalists is that the partial vacuum in the supply and demand cycle will inevitably be met by ecologically unstable developments in tropi­cal regions to establish monoculture plantations of “energy crops” to further harden the international trade in “off-the-shelf” biofuels.

In the short term, it can readily be concluded that all the elements needed to establish a cellulosic ethanol industry do now exist — and have existed for a decade (and possibly longer) — and that the biotechnologists could now leave the stage to determined commercial interests to finance, set up, and maintain industrial pro­duction facilities. Casualties are inevitable: some second-generation companies will undoubtedly fail financially, but this will be a part of whatever learning curve the new biobased industry will encounter, and as a safety net, in the United States and other OECD nations, enough government support is likely to remain on offer to smooth the transition as long as crude oil prices are sufficiently high for biofuels to be pump price competitive with gasoline.

The commercialization of GM technologies for staple food crops such as wheat has faced the obstacles of social and market opposition and resistance.313 Fortunately (or fortuitously), gene transformation techniques for improving wheat yield have been challenging for plant biotechnology: bread wheat (Triticum aestivum L.) has one of the largest and most complex plant polyploid genomes, 80% of which consists of non­coding sequences, deriving from three ancestral genomes; new initiatives to analyze the minority expressed portion of the wheat genome are ongoing, but the structural complexity of the genome has been of enormous value to agronomy over millennia because major chromosomal rearrangements and deletions are well tolerated.314

Public perceptions may be more favorable to GM technologies applied to dedi­cated energy crops, although even here the fear is that of the introduction and spread of unwanted genes into natural populations with consequences that are difficult to accurately predict. Geographical isolation of energy crops (as of GM plants designed to synthesize high-value biopharmaceuticals) is one extreme solution but flies in the face of the limited land availability for nonfood crops. Even if the entire U. S. corn and soybean crops were to be devoted to bioethanol and biodiesel production (with complete elimination of all direct and indirect food uses), only 5.3% of the 2005 gasoline and diesel requirement could be met.315 Expressed another way, an area of land nearly 20-fold larger than that presently used for corn and soybean cultivation would be required for bioethanol and biodiesel energy crops. Concerns relating to land delineation are highly probable even if a wheat straw/corn stover bioeconomy is used as the main supply of feedstocks for bioethanol production.316

Equally inevitable, however, is that GM approaches will be applied to either dual food-energy crops or to dedicated energy crops such as fast-growing willow and switchgrass. The USDA-ARS Western Regional Research Center, Albany, Califor­nia, has created a gene inventory of nearly 8,000 gene clusters in switchgrass, 79% of which are similar to known protein or nucleotide sequences.317 A plasmid system has also been developed for the transformation of switchgrass with a herbicide resistance gene.318 Grasses share coding sequences of many of their genes: the sugarcane genome shows an 81.5% matching frequency with the rice genome and even a 70.5% matching frequency with the genome of the “lower” plant, Arabidopsis thaliana.319 The risk of gene transfer across species and genera in the plant kingdom is, therefore, very real.

A selection of patents relevant to transgenic crops and other plant genetic manip­ulations for bioethanol production is given in table 4.5.

Of the biofuels discussed in this section, only biodiesel has reached full com­mercialization and not all of the others have even been tested in pilot plants and demonstration units as a prelude to scale up. Any conclusions as to their impact on fuel usage, the substitution of fossil fuels or reduction in greenhouse gas emissions is consequently highly speculative. Nevertheless, a European consortium of research institutes and university groups from five nations has commenced building advanced biofuels into a comprehensive model of biofuel chain options until 2030, commis­sioned by the European Union to identify a robust biofuels strategy to minimize costs and identify key technological, legislative, and policy developments that are required.140 Among the key preliminary predictions are that:

• Biodiesel and cereal — and sugar-derived bioethanol use in Europe will peak at approximately 10% of total transportation fuels by 2020.

• By 2020, advanced biofuels (including lignocellulosic ethanol) will con­tribute an equal share of total transportation fuels but will peak at no more than 30% by 2050, the maximum being limited by the amount of land avail­able for energy crops.

• The introduction of advanced biofuel options may meet considerable intro­ductory cost barriers.

• Advanced biofuels may require “stepping-stone” strategies (e. g., the short­term development of lignocellulosic biomass supply chains for power gen­eration by cofiring in power generating plants) or realistic synergies (e. g., coproduction of FT diesel and hydrogen for use in fuel cells).

• Commencing by 2020, the use of hydrogen-powered fuel cells is the only route to replacing fossil fuels for 50% or more of light-duty vehicle trans­portation needs.

• For heavy duty trucks, where fuel cells are unlikely to meet the demands for either high continuous loads or in long-distance transport, advanced biofuels may be the long-term market solution.

That biofuels — in particular, ethanol and biodiesel — might only be a transi­tional rather than a lasting solution for sustainable passenger transport is one chal­lenging hypothesis but one with a rival: that globalization of biofuels will lead to a split between the North (the United States, Europe, and Japan) and the South (Latin America, Africa, and South and Southeastern Asia), the former increas­ingly focused on the production of advanced biofuels (including hydrogen-based systems), whereas the South develops a long-term economic strategy based on sugar ethanol and biodiesel, trading internationally and gradually supplanting OPEC.141 Tropical countries “do it better,” that is, can produce ethanol with positive energy gains, because they achieve at least twice the yield per hectare than can be dem­onstrated in temperate countries producing corn — or grain-derived ethanol; as loca­tions for biodiesel crops, locales in the Southern Hemisphere simply cannot be bettered, for land availability, climate, the type of plant species grown or the yields extracted from them — table 6.7 lists recent investment programs in the Southern Hemisphere.142

In this analysis, the status of India and China is ambiguous, straddling the bound­ary between agricultural and industrial nations: both rank in the top 11 of nations with the least car ownership (6 and 11/1000 population, respectively), but both are in

• Innovative heterogeneous catalysts for transesterification reactions in biodiesel production144

• Producing very high quality FT diesel (from fossil sources) to be used in regions with very stringent specifications for diesel fuels and their emis­sions or to upgrade below specification diesels by blending145

• The utilization of (perhaps, unpurified) biomass-generated syngas in fer­mentations to support the production of either ethanol or major commod­ity chemicals146,147

The existing scientific base in OECD economies is, however, most likely to be best employed by successfully defining novel technologies to tackle the dual problems of dwindling oil reserves and out-of-control CO2 emissions. Beyond the second-genera­tion biofuels discussed in this chapter lies a raft of long-considered (sometimes, long — researched) topics, to which — to differing extents — the adjective “speculative” is often applied. All of these have strong biotechnological inputs and can be grouped as the radical options for biofuels.

European commentators and analysts were far less sanguine on the desirability of fuel ethanol as a strategic industry for the future in the mid-1980s. This was a time of steeply falling oil prices, both expressed in real terms and in the actual selling price (figure 1.3). In 1987, two independent assessments of ethanol production from agricultural feed­stocks were published in the United Kingdom and Europe.17-19 Across Europe, the introduction of lead-free fuels heralded an important new market for ethanol and other additives but the competition among these compounds (including MTBE and metha­nol) was likely to be intense for the estimated 2-million-tonnes/year market by 1998.17

The U. K. survey included wheat grain and sugarbeet as possible local sources of carbohydrates; in both cases, raw plant materials dominated the production cost analysis (table 5.6). The monetary value of coproducts were important, although only animal feeds were considered as viable sources of income to offset ethanol production

costs.17 The market prices for all major agricultural products were determined by the price support policies of the Common Agricultural Policy (CAP), an essential part of the Treaty of Rome (March, 1957), under which the European Economic Community (EEC) was set up and regulated; among its many provisions, the CAP was designed to ensure both a fair standard of living for farmers and reasonable consumer prices, and the CAP operated to guarantee a minimum price for basic agricultural products through intervention prices and protected the community’s internal markets against fluctuations in world prices through the establishment of threshold prices. Technical progress was, however, also a goal of the CAP to increase agricultural productivity. The CAP has been controversial inside the EEC and subsequently the European Community and European Union as individual member states have received varying benefits from the policy but it has the advantage of enabling commodity prices to be more predictable, a useful factor when calculating possible trends in feedstock prices for the production of biofuels. In 1987, the likely costs of ethanol from wheat and sugarbeet were greatly in excess of the refinery price of petrol (gasoline), with a cost ratio of 3.2-4.4:1, allowing for the lower energy content of ethanol.17 The continuing influence of the CAP was moreover highly unlikely to reduce feedstock costs for bioethanol to be price competitive with conventional fuels.

The second volume of the U. K. study gave outline production cost summaries for ethanol derived from wood (no species was specified) using acid and enzymatic hydrolysis for the liberation of glucose from cellulose (table 5.7). Additionally, straw residues were considered from cereals (wheat, barley, and oats), field beans, and oil seed rape (canola) using acid and enzymic hydrolysis; electricity and lignin were modeled as saleable coproducts. No source for ethanol could yield a product with a production cost less than three times that of conventional fuels (figure 5.4). These poor economics resulted in the authors being unable to recommend initiat­ing a large program of work directed toward bioethanol production in the United Kingdom, although continued support of existing research groups was favored to enable the United Kingdom to be able to take advantage of fundamental break­throughs, especially in lignocellulose conversion. A return to the high oil prices experienced in 1973-4 and 1978-80 was considered unlikely until well into the

twenty-first century in any case, because transport costs were inevitably an impor­tant element of feedstock costs in the United Kingdom, rising oil prices would tend to increase the total costs for bioethanol production.18

The European study presented less detailed economic data but considered a wider range of feedstocks, not all of which were (or are) major agricultural products across the whole of Europe, but which represented potential sources for expanded agricultural production or as dedicated energy crops. For production facilities capable
of manufacturing more than 150 million liters (40 million gallons) of ethanol/year, wheat grain was the cheapest source with a production cost of the feedstock equiva­lent to $0.36/gallon (converting the now obsolete European currency unit values to U. S. dollar at the exchange rate prevailing in early January 1987), followed by corn ($0.62/gallon), sugarbeet ($0.83/gallon), Jerusalem artichoke ($0.87/gallon), potatoes ($1.89/gallon), and wine[48] ($2.73/gallon).19 The estimated cost of ethanol production from wheat was 0.49 ECU/l (equivalent to 530/l, or $2.01/gallon); in comparison, the Rotterdam refinery price for premium gasoline in late 1986 was approximately 100/l. The consultants who assembled the report concluded:

1. Encouragement of a bioethanol program was not in the economic interests of the European Community

2. A large reduction of the feedstock costs would be required to show a net economic benefit from bioethanol production

3. Alternatively, an oil price in the range $30-40/barrel would be required to achieve economic viability for bioethanol

Although the conversion of ethanol to ethylene was technically feasible, a cost analy­sis of this route indicated that it would be even less viable economically than ethanol production as a fuel additive.

The European study included an analysis of the development of fuel ethanol indus­tries in Brazil and the United States, noting that the bulk of the financial incentives in the U. S. corn ethanol sector benefited the large producers rather than the small opera­tors or the corn farmers; even more disturbing to European decision makers was the conclusion that blenders had benefited disproportionately, enjoying effectively cost — free ethanol as a gasoline additive during 1986, and using the available subsidies to start a price-cutting war between ethanol producers rather than promoting total sales. The prospect of subsidies being essential for establishing and maintaining a bioetha­nol program in Europe was nevertheless consistent with the European Community and its long-established strategic approach to agricultural development.

Hydrogen ions (protons, H+) can accept reducing equivalents (conventionally represented as electrons, e-) generated either photosynthetically or by the oxidation of organic and inorganic substrates inside microbial cells:

2e — + 2H+ ^ H2

The terminal electron donor (e. g., reduced ferredoxin) could donate electrons to the anode of a battery. Protons could then, in the presence of O2, complete the electric circuit at the cathode by the reaction:

O2 + 4e — + 4H+ ^ 2H2O

thus forming a highly environmentally friendly source of electric power (a battery), fueled by microbial metabolic activity. That, in essence, is the definition of a microbial fuel cell (MFC).98-100

At its simplest, an MFC is a dual-chamber device with an electrolyte, a cation exchange membrane to separate anodic and cathodic compartments, a supply of O2 for the cathode, and an optional sparge of inert gas for the anode (figure 1.11). The transfer of electrons to the anode may be either direct (via unknown terminal electron donors on the cell surface) or employing redox-active “mediators” that can be reduced by the cells and reoxidized at the anode (e. g., neutral red reduced by hydrogenase).101,102 A wide spectrum of microbial species have been tested in MFC environments, usually anaerobes or facultative anaerobes chosen to function in the O2-deficient anode compartment. Recent examples include

• Immobilized cells of the yeast Hansenula anomala103

• A mixed microbial community of Proteobacterium, Azoarcus, and Desulfuromonas species with ethanol as the fuel source104

• Desulfitobacterium hafniense with humic acids or the humate analog anthraquinone-2,6-disulfonate added as an electron-carrying mediator with formic acid, H2, lactate, pyruvate, or ethanol as the fuel105

• E. coli in MFCs as power sources for implantable electronic devices106

The first use of the term “microbial fuel cell” appears to date from the early 1960s in studies of hydrocarbon-metabolizing Nocardia bacteria by research scientists of the Mobil Oil Company, but the basic concepts may date back 30 or even 50 years earlier.101 Developments of MFCs as commercial and industrial functional­ities are meth

y these systems is currently limited, primarily by high internal (ohmic) resistance, but improvements in system architecture might result in power generation that is more dependent on the bioenergetic capabilities of the

Cathodic compartment

FIGURE 7.11 Redox reactions occurring in an MFC: MED is the soluble mediator reduced by the microbial terminal electron donor at the microbial cell surface.

microorganisms.108 They are close to devices mobilized for the sets of science fiction films that call on “bioelectricity” in all its many conceptual forms (e. g., in the Matrix trilogy) and can easily be imagined in self-reliant ecosystems in deep space travel (Silent Running). But are they meaningful additions to the biofuels armory on planet Earth?

Creating a scalable architecture for MFCs is essential to provide large surface areas for oxygen reduction at the cathode and bacteria growth on the anode; a tubular ultrafiltration membrane with a conductive graphite coating and a nonprecious metal catalyst can be used to produce power in an MFC and is a promising architecture that is intrinsically scalable for creating larger systems.109 For the anodes, highly conduc­tive noncorrosive materials are needed that have a high specific surface area (i. e., surface area per volume) and an open structure to avoid biofouling; graphite fiber brush anodes have high surface areas and a porous structure can produce high power densities, qualities that make them ideal for scaling up MFC systems.110 The technol­ogy has long existed for very-large-scale microbial fermentations for bulk chemicals (e. g., citric acid and amino acids), with stirred tank volumes greater than 500,000 l being widely used; such structures could, in principle, be the “anodic” compartments of stationary MFC generators. Because rumen bacteria have been shown to generate electricity from cellulosic materials, potentially immense substrate supplies could be available for MFC arrays.111 Even greater flexibility can be designed, for example, coproducing H2 and ethanol production from glycerol-containing wastes discharged from a biodiesel fuel production plant with Enterobacter aerogenes in bioelectro­chemical cells with thionine as the exogenous electron transfer mediator.112

Cellulases are known to be adsorbed by lignins, thus reducing their catalytic potential for cellulose digestion.139 140 Steam pretreatment may even increase the potential cellulase-binding properties of lignin.141 Enzymatic removal of lignin residues from pretreated lignocellulosic materials has received little attention. Laccase (p-diphenol:O2 oxidoreductase, EC 1.10.3.2) catalyzes the cleavage of C-C and C-ether bonds in lignins.142 Laccase treatment of steam-pretreated softwoods was shown to improve subsequent cellulase-mediated saccharification.143 The second major class of lignin-degrading enzymes, peroxidases, includes many additional targets for enzyme manufacturers to produce and test as adjuncts to cellulases for the digestion of pretreated lignocellulosic materials.144

Coating the surfaces of lignin residues in pretreated lignocellulosic materials by adding a protein before cellulase incubation is an extension of a long-practiced biochemist’s technique known as “sacrificial protein” use to protect sensitive enzymes. Adding a commercially available protein, bovine serum albumin, to corn stover pretreated with either dilute sulfuric acid or ammonia fiber explosion and to Douglas fir pretreated by SO2 steam explosion increases cellulose saccharification and is a promising strategy to reduce enzyme requirements for feedstock processing if a suitable source of low-cost protein is identified.145

Many of the problems encountered in regulating both growth and metabolism in actively growing, high-cell-density systems could, in principle, be avoided by immobilizing the producer cells in or on inert supports and reusing them (if their viability can be maintained) for repetitive cycles of production. This is akin to trans­forming whole-cell biocatalysis to a more chemically defined form known for more than a century in the fine chemicals manufacturing industry. Cost savings in the power inputs required to agitate large-volume fermentors (100,000-500,000 l), in their cooling and their power-consuming aeration, spurred the fermentation industry at large to consider such technologies, and serious attempts to immobilize productive ethanologens began in the 1970s.

Alginate beads, prepared from the hydrophilic carbohydrate polymer alginic acid (extracted from kelp seaweed), are porous, compatible with an aqueous environ­ment, and could be loosely packed; in such a packed-bed bioreactor, perfused with a nutrient and substrate solution, a high volumetric productivity of ethanol produc­tion could be maintained for up to 12 days with a less than 10% loss of productive capacity.175 With cells of the yeast Kluyveromyces marxianus supplied with a Jeru­salem artichoke tuber extract, the maximum volumetric productivity was 15 times than for a conventional stirred tank fermentor. With S. cerevisiae in a fluidized bed contained in a closed circuit, ethanol production was possible with glucose solutions of up to 40% w/v at a relatively low temperature (18°C).176

As with continuous fermentation technologies, potable-alcohol manufacturers expressed interest in and actively developed demonstration facilities for immobi­lized yeast cells but failed to fully exploit the potential of the innovations — again because the product failed to meet organoleptic and other exacting specifications for the industry.177 Many different matrices were investigated beyond alginate and other seaweed polymers, including ceramic materials, synthetic polymers, and stainless steel fibers, to adsorb or entrap cells in up to five different reactor geometries:

1. The packed bed, the simplest arrangement and capable of upward or down­ward flow of the liquid phase

2. The fluidized bed, where mixing of gas, liquid, and solid phases occurs continuously

3. Airlift and bubble column bioreactors

4. Conventional stirred tank reactors

5. Membrane bioreactors where the cells are free but retained by a semiper­meable membrane

Beer producers have adopted immobilized cells to selectively remove unwanted aroma compounds from the primary fermentation product, “green” beer, and to both produce and remove aldehydes from alcohol-free and low-alcohol beers using strains and mutants of S. cerevisiae.178 For the main fermentation, however, immobilized cells remain a “promising” option for further development, but the added cost of immobilization techniques are impossible to justify for commercial processes with­out much stronger increases in volumetric productivity.

Cost-effective options have been explored for fuel ethanol production. In Brazil, improved fermentation practices and efficiencies have taken up the slack offered by low-intensity production system, and further progress requires technological innovations; immobilizing yeast ethanologens on sugarcane stalks is an eminently practical solution to providing an easily sourced and import-substituting support matrix:179 [39]

A summary of data from other studies of immobilized cells relevant to fuel ethanol production is given in table 4.2.180-184 The liquid phase can be recirculated in an immobilized system to maximize the utilization of the fermentable sugars; this is particularly useful to harmonize attaining maximal productivity (grams of ethanol produced per liter per hour) and maximum ethanol yield (as a percentage of the maximum theoretically convertible), with these optima occurring at different rates of flow with bacterial and yeast ethanologens.180

Among other recent developments, recombinant xylose-utilizing Z. mobilis cells were immobilized in photo-cross-linked resins prepared from polyethylene or poly­propylene glycols and shown to efficiently utilize both glucose and xylose from acid hydrolysates of cedar tree wood, rice straw, newspaper, and bagasse — unfortunately, the pretreatment was unfashionable, that is, a concentrated sulfuric process, and dif­ficult to precisely relate to modern trends in lignocellulosic processes.185 Alginate — immobilized S. cerevisiae cells have also been used in conjunction with a five-vessel cascade reactor in a continuous alcohol fermentation design.186 Sweet sorghum is a major potential source of fuel ethanol production in China where laboratory studies have progressed immobilized yeast cells to the stage of a 5-l bioreactor with stalk juice (i. e., equivalent to pressed cane sugar juice) as a carbon substrate.187

Insofar as immobilized cells represent a stationary phase or a population of very slowly growing cells, they may exhibit enhanced resistance to growth inhibitors and other impurities in substrate solutions that are more injurious to actively dividing cells. Immobilized cells of various microbial species, for example, are considered to be more resistant to aromatic compounds, antibiotics, and low pH, whereas immo­bilized S. cerevisiae is more ethanol-tolerant.178 This opens the novel possibility that immobilized yeast could be used to ferment batches of microbial toxin-containing feedstocks: a trichothecene mycotoxin inhibits protein synthesis and mitochon­drial function in S. cerevisiae and at 200 mg/l can cause cell death; conversely, fermentation is not affected and glucose metabolism may be positively redirected toward ethanol production, suggesting that trichothecene-contaminated grain could be “salvaged” for fuel ethanol manufacture.188 Alginate-encapsulated yeast cells are certainly able to withstand toxic sugar degradation products in acid hydrolysates prepared from softwoods (mainly spruce wood); such immobilized cells could fully ferment the glucose and mannose sugars within 10 hr, whereas free cells could not accomplish this within 24 hours, although the encapsulated cells lost their activity in subsequent batch fermentations.189

When will the oil run out? Various estimates put this anywhere from 20 years from now to more than a century in the future. The shortfall in energy might eventually be made up by developments in nuclear fusion, fuel cells, and solar technologies, but what can substitute for gasoline and diesel in all the internal combustion engine — powered vehicles that will continue to be built worldwide until then? And what will stand in for petrochemicals as sources of building blocks for the extensive range of “synthetics” that became indispensable during the twentieth century?

Cellulose — in particular, cellulose in “lignocellulosic biomass” — embodies a great dream of the bioorganic chemist, that of harnessing the enormous power of nature as the renewable source for all the chemicals needed in a modern, bioscience — based economy.1 From that perspective, the future is not one of petroleum crackers and industrial landscapes filled with the hardware of synthetic organic chemistry, but a more ecofriendly one of microbes and plant and animal cells purpose-dedicated to the large-scale production of antibiotics and blockbuster drugs, of monomers for new biodegradable plastics, for aromas, fragrances, and taste stimulators, and of some (if not all) of the novel compounds required for the arrival of nanotechnologies based on biological systems. Glucose is the key starting point that, once liberated from cellulosic and related plant polymers, can — with the multiplicity of known and hypothesized biochemical pathways in easily cultivatable organisms — yield a far greater multiplicity of both simple and complex chiral and macromolecular chemical entities than can feasibly be manufactured in the traditional test tube or reactor vessel.

A particular subset of the microbes used for fermentations and biotransforma­tions is those capable of producing ethyl alcohol — ethanol, “alcohol,” the alcohol whose use has both aided and devastated human social and economic life at various times in the past nine millennia. Any major brewer with an international “footprint” and each microbrewery set up to diversify beer or wine production in contention with those far-reaching corporations use biotechnologies derived from ancient times, but that expertise is also implicit in the use of ethanol as a serious competitor to gasoline in automobile engines. Hence, the second vision of bioorganic chemists has begun to crystallize; unlocking the vast chemical larder and workshop of natu­ral microbes and plants has required the contributions of microbiologists, microbial physiologists, biochemists, molecular biologists, and chemical, biochemical, and metabolic engineers to invent the technologies required for industrial-scale produc­tion of “bioethanol.”

The first modern social and economic “experiment” with biofuels — that in Brazil — used the glucose present (as sucrose) in cane sugar to provide a readily available and renewable source of readily fermentable material. The dramatic rise in oil prices in 1973 prompted the Brazilian government to offer tax advantages to those who would power their cars with ethanol as a fuel component; by 1988, 90% of the cars on Brazilian roads could use (to varying extents) ethanol, but the collapse

in oil prices then posed serious problems for the use of sugar-derived ethanol. Since then, cars have evolved to incorporate “dual-fuel” engines that can react to fluctua­tions in the market price of oil, Brazilian ethanol production has risen to more than 16 million liters/year, and by 2006, filling up with ethanol fuel mixes in Brazil cost up to 40% less than gasoline.

Sugarcane thrives in the equatorial climate of Brazil. Further north, in the mid­western United States, corn (maize, Zea mays) is a major monoculture crop; corn accumulates starch that can, after hydrolysis to glucose, serve as the substrate for eth­anol fermentation. Unlike Brazil, where environmentalists now question the destruc­tion of the Amazonian rain forest to make way for large plantations of sugarcane and soya beans, the Midwest is a mature and established ecosystem with high yields of corn. Cornstarch is a more expensive carbon substrate for bioethanol production, but with tax incentives and oil prices rising dramatically again, the production of ethanol for fuel has become a significant industry. Individual corn-based ethanol production plants have been constructed in North America to produce up to 1 million liters/day, and in China 120,000 liters/day, whereas sugarcane molasses-based facilities have been sited in Africa and elsewhere.2

In July 2006, the authoritative journal Nature Biotechnology published a cluster of commentaries and articles, as well as a two-page editorial that, perhaps uniquely, directed its scientific readership to consult a highly relevant article (“Ethanol Frenzy”) in Bloomberg Markets. Much of the discussion centered on the economic viability of fuel ethanol production in the face of fluctuating oil prices, which have inhibited the development of biofuels more than once in the last half century.3 But does bioethanol production consume more energy than it yields?4 This argument has raged for years; the contributors to Nature Biotechnology were evidently aware of the controversy but drew no firm conclusions. Earlier in 2006, a detailed model-based survey of the economics of corn-derived ethanol production processes concluded that they were viable but that the large-scale use of cellulosic inputs would better meet both energy and environmental goals.5 Letters to the journal that appeared later in the year reiter­ated claims that the energy returns on corn ethanol production were so low that its production could only survive if heavily subsidized and, in that scenario, ecological devastation would be inevitable.6

Some energy must be expended to produce bioethanol from any source — in much the same way that the pumping of oil from the ground, its shipping around the world, and its refining to produce gasoline involves a relentless chain of energy expenditure. Nevertheless, critics still seek to be persuaded of the overall benefits of fuel ethanol (preferring wind, wave, and hydroelectric sources, as well as hydrogen fuel cells). Meanwhile its advocates cite reduced pollution of the atmosphere, greater use of renewable resources, and erosion of national dependence on oil imports as key factors in the complex overall cost-benefit equation.

To return to the “dream” of cellulose-based chemistry, there is insufficient arable land to sustain crop-based bioethanol production to more than fuel-additive levels worldwide, but cellulosic biomass grows on a massive scale — more than 7 x 1010 tons/year — and much of this is available as agricultural waste (“stalks and stems”), forestry by-products, wastes from the paper industry, and as municipal waste (card­board, newspapers, etc.).7 Like starch, cellulose is a polymeric form of glucose; unlike starch, cellulose cannot easily be prepared in a highly purified form from many plant sources. In addition, being a major structural component of plants, cellulose is com­bined with other polymers of quite different sugar composition (hemicelluloses) and, more importantly, with the more chemically refractive lignin. Sources of lignocel — lulosic biomass may only contain 55% by weight as fermentable sugars and usually require extensive pretreatment to render them suitable as substrates for any microbial fermentation, but that same mixture of sugars is eminently suitable for the produc­tion of structures as complex as aromatic intermediates for the chemical industry.8

How practical, therefore, is sourcing lignocellulose for bioethanol production and has biotechnology delivered feasible production platforms, or are major develop­ments still awaited? How competitive is bioethanol without the “special pleading” of tax incentives, state legislation, and (multi)national directives? Ultimately, because the editor of Nature Biotechnology noted that, for a few months in 2006, a collection of “A-list” entrepreneurs, venture capitalists, and investment bankers had promised $700 million to ethanol-producing projects, the results of these developments in the real economy may soon refute or confirm the predictions from mathematical mod — els.9 Fiscal returns, balance sheets, and eco audits will all help to settle the major issues, thus providing an answer to a point made by one of the contributors to the flurry of interest in bioethanol in mid-2006: “biofuels boosters must pursue and pro­mote this conversion to biofuels on its own merits rather than by overhyping the rela­tive political, economic and environmental advantages of biofuels over oil.”10

Although the production of bioethanol has proved capable of extensive scale up, it may be only the first — and, by no means, the best — of the options offered by the biological sciences. Microbes and plants have far more ingenuity than that deduced from the study of ethanol fermentations. Linking bioethanol production to the syn­thesis of the bioorganic chemist’s palette of chemical feedstocks in “biorefineries” that cascade different types of fermentations, possibly recycling unused inputs and further biotransforming fermentation outputs, may address both financial and envi­ronmental problems. Biodiesel (simple alkyl esters of long-chain fatty acids in veg­etable oils) is already being perceived as a major fuel source, but further down the technological line, production of hydrogen (“biohydrogen”) by light-driven or dark fermentations with a variety of microbes would, as an industrial strategy, be akin to another industrial revolution.11

A radically new mind-set and a heightened sense of urgency were introduced in September 2006 when the state of California moved to sue automobile manufactur­ers over tailpipe emissions adding to atmospheric pollution and global warming. Of the four major arguments adduced in favor of biofuels — long-term availability when fossil fuels become depleted, reduced dependence on oil imports, develop­ment of sustainable economies for fuel and transportation needs, and the reduction in greenhouse gas emissions — it is the last of these that has occupied most media attention in the last three years.12 In October 2006, the first quantitative model of the economic costs of not preventing continued increases in atmospheric CO2 pro­duced the stark prediction that the costs of simply adapting to the problems posed by global warming (5-20% of annual global GDP by 2050) were markedly higher than those (1% of annual global GDP) required to stabilize atmospheric CO2.13 Although developing nations will be particularly hard hit by climate changes, industrialized

nations will also suffer economically as, for example, rising sea levels require vastly increased flood defense costs and agricultural systems (in Australia and elsewhere) become marginally productive or collapse entirely.

On a more positive note, the potential market offered to technologies capable of reducing carbon emissions could be worth $500 billion/year by 2050. In other words, while unrestrained increase in greenhouse gas emissions will have severe consequences and risk global economic recession, developing the means to enable a more sustainable global ecosystem would accelerate technological progress and establish major new industrial sectors.

In late 2007, biofueled cars along with electric and hybrid electric-gasoline and (in South America and India) compressed natural gas vehicles represented the only immediately available alternatives to the traditional gasoline/internal combustion engine paradigm. Eventually, electric cars may evolve from a niche market if renew­able energy sources expand greatly and, in the longer term, hydrogen fuel cells and solar power (via photovoltaic cells) offer “green” vehicles presently only known as test or concept vehicles. The International Energy Agency estimates that increasing energy demand will require more than $20 trillion of investment before 2030; of that sum, $200 billion will be required for biofuel development and manufacture even if (in the IEA’s assessments) the biofuels industry remains a minor contributor to trans­portation fuels globally.14 Over the years, the IEA has slowly and grudgingly paid more attention to biofuels, but other international bodies view biofuels (especially the second-generation biofuels derived from biomass sources) as part of the growing family of technically feasible renewable energy sources: together with higher-effi­ciency aircraft and advanced electric and hybrid vehicles, biomass-derived biofuels are seen as key technologies and practices projected to be in widespread use by 2030 as part of the global effort to mitigate CO2-associated climate change.15

In this highly mobile historical and technological framework, this book aims to analyze in detail the present status and future prospects for biofuels, from ethanol and biodiesel to biotechnological routes to hydrogen (“biohydrogen”). It emphasizes ways biotechnology can improve process economics as well as facilitate sustainable agroindustries and crucial elements of the future bio-based economy, with further innovations required in microbial and plant biotechnology, metabolic engineering, bioreactor design, and the genetic manipulation of new “biomass” species of plants (from softwoods to algae) that may rapidly move up the priority lists of funded research and of white (industrial biotech), blue (marine biotech), and green (environ­mental biotech) companies.

A landmark publication for alternative fuels was the 1996 publication Hand­book on Bioethanol: Production and Utilization, edited by Charles E. Wyman of the National Renewable Energy Laboratory (Golden, Colorado). That single-volume, encyclopedic compilation summarized scientific, technological, and economic data and information on biomass-derived ethanol (“bioethanol”). While highlighting both the challenges and opportunities for such a potentially massive production base, the restricted use of the “bio” epithet was unnecessary and one that is now (10 years later) not widely followed.16 Rather, all biological production routes for ethanol — whether from sugarcane, cornstarch, cellulose (“recycled” materials), lignocellulose (“biomass”), or any other nationally or internationally available plant

source — share important features and are converging as individual producers look toward a more efficient utilization of feedstocks; if, for example, sugarcane-derived ethanol facilities begin to exploit the “other” sugars (including lignocellulosic com­ponents) present in cane sugar waste for ethanol production rather than only sucrose, does that render the product more “bio” or fully “bioethanol”?

As the first biofuel to emerge into mass production, (bio)ethanol is discussed in chapter 1, the historical sequence being traced briefly from prehistory to the late nineteenth century, the emergence of the petroleum-based automobile industry in the early twentieth century, the intermittent interest since 1900 in ethanol as a fuel, leading to the determined attempts to commercialize ethanol-gasoline blends in Brazil and in the United States after 1973. The narrative then dovetails with that in Handbook on Bioethanol: Production and Utilization, when cellulosic and lig- nocellulosic substrates are considered and when the controversy over calculated energy balances in the production processes for bioethanol, one that continued at least until 2006, is analyzed. Chapters 2, 3, and 4 then cover the biotechnol­ogy of ethanol before the economics of bioethanol production are discussed in detail in chapter 5, which considers the questions of minimizing the social and environmental damage that could result from devoting large areas of cultivatable land to producing feedstocks for future biofuels and the sustainability of such new agroindustries.

But are bioethanol and biodiesel (chapter 6) merely transient stopgaps as trans­portation fuels before more revolutionary developments in fuel cells usher in biohy­drogen? Both products now have potential rivals (also discussed in chapter 6). The hydrogen economy is widely seen as providing the only workable solution to meet­ing global energy supplies and mitigating CO2 accumulation, and the microbiology of “light” and “dark” biohydrogen processes are covered (along with other equally radical areas of biofuels science) in chapter 7. Finally, in chapter 8, rather than being considered as isolated sources of transportation fuels, the combined production of biofuels and industrial feedstocks to replace eventually dwindling petrochemicals — in “biorefineries” capable of ultimately deriving most, if not all, humanly useful chemicals from photosynthesis and metabolically engineered microbes — rounds the discussion while looking toward attainable future goals for the biotechnologists of energy production in the twenty-first century, who very possibly may be presented with an absolute deadline for success.

For to anticipate the answer to the question that began this preface, there may only be four decades of oil left in the ground. The numerical answer computed for this shorter-term option is approximately 42 years from the present (see Figure 5.13 in chapter 5) — exactly the same as the answer to the ultimate question of the uni­verse (and everything else) presented in the late 1970s by the science fiction writer Douglas Adams (The Hitchhiker’s Guide to the Galaxy, Pan Books, London). The number is doubly unfortunate: for the world’s senior policy makers today, agreement (however timely or belated) on the downward slope of world oil is most likely to occur well after their demise, whereas for the younger members of the global popu­lation who might have to face the consequences of inappropriate actions, misguided actions, or inaction, that length of time is unimaginably distant in their own human life cycles.

Four decades is a sufficiently long passage of time for much premier quality sci­entific research, funding of major programs, and investment of massive amounts of capital in new ventures: the modern biopharmaceutical industry began in the early 1980s from a scattering of research papers and innovation; two decades later, biotech companies like Amgen were dwarfing long-established pharmaceutical multination­als in terms of income stream and intellectual property.

But why (in 2008) write a book? When Jean Ziegler, the United Nations’ “inde­pendent spokesman on the right to food,” described the production of biofuels as a “crime against humanity” and demanded a five-year moratorium on biofuels pro­duction so that scientific research could catch up and establish fully the methods for utilizing nonfood crops, he was voicing sentiments that have been gathering like a slowly rising tide for several years.17 Precisely because the whole topic of biofu­els — and especially the diversion of agricultural resources to produce transporta­tion fuels, certainly for industry, but also for private motorists driving vehicles with excellent advertising and finance packages but woefully low energy efficiencies — is so important, social issues inevitably color the science and the application of the derived technology. Since the millennium, and even with rocketing oil prices, media coverage of biofuels has become increasingly negative. Consider the following selection of headlines taken from major media sources with claims to international readerships:

Biofuel: Green Savior or Red Herring? (CNN. com, posted April 2, 2007) Biofuels: Green Energy or Grim Reaper? (BBC News, London, September 22, 2006)

Scientists Are Taking 2nd Look at Biofuels (International Herald Tribune, January 31, 2007)

Green Fuel Threatens a ‘Biodiversity Heaven’ (The Times, London, July 9, 2007) Biofuel Demand to Push Up Food Prices (The Guardian, London, July 5, 2007) Plantation Ethanol ‘Slaves’ Freed (The Independent, London, July 5, 2007)

The Biofuel Myths (International Herald Tribune, July 10, 2007)

Biofuel Gangs Kill for Green Profits (The Times, London, June 3, 2007)

Dash for Green Fuel Pushes Up Price of Meat in US (The Times, London, April 12, 2007)

The Big Green Fuel Lie (The Independent, London, March 5, 2007)

How Biofuels Could Starve the Poor (Foreign Affairs, May/June 2007)

Biofuel Plant ‘Could Be Anti-Green’ (The Scotsman, Edinburgh, July 5, 2007)

To Eat… or to Drive? (The Times, London, August 25, 2007)

These organizations also carry (or have carried) positive stories about biofuels (“The New Gold Rush: How Farmers Are Set to Fuel America’s Future” or “Poison Plant Could Help to Cure the Planet,”18) but a more skeptical trend emerged and hardened during 2006 and 2007 as fears of price inflation for staple food crops and other concerns began to crystallize. In the same week in August 2007, New Zealand began its first commercial use of automobile bioethanol, whereas in England, the major long­distance bus operator abandoned its trials of biodiesel, citing environmental damage and unacceptable diversion of food crops as the reasons. On a global ecological basis, plantations for biofuels in tropical regions have begun to be seriously ques­tioned as driving already endangered wildlife species to the edge of oblivion.

Perhaps most damning of all, the “green” credentials of biofuels now face an increasing chorus of disbelief as mathematical modeling erodes the magnitudes of possible benefits of biofuels as factors in attempts to mitigate or even reverse greenhouse gas emissions — at its most dramatic, no biofuel production process may be able to rival the CO2-absorbing powers of reforestation, returning unneeded croplands to savannah and grasslands.19 The costs of biofuels escalate, whereas the calculated benefits in reducing greenhouse gas emissions fall.20 The likely impact of a burgeoning world trade in biofuels — and the subject already of highly vocal complaints about unfair trade practices — on the attainment of environmental goals in the face of economic priorities21 is beginning to cause political concern, especially in Europe.22

But why write a book? The Internet age has multiple sources of timely information (including all the above-quoted media stories), regularly updated, and available 24/7. The thousands of available sites offer, however, only fragmentary truths: most are campaigning, selective in the information they offer, focused, funded, targeting, and seeking to persuade audiences or are outlets for the expression of the views and visions of organizations (“interested parties”). Most academic research groups active in bio­fuels also have agendas: they have intellectual property to sell or license, genetically engineered microbial strains to promote, and results and conclusions to highlight in reviews. This book is an attempt to broaden the discussion, certainly beyond bioetha­nol and biodiesel, placing biofuels in historical contexts, and expanding the survey to include data, ideas, and bioproducts that have been visited at various times over the last 50 years, a time during which widely volatile oil prices have alternately stimu­lated and wrecked many programs and initiatives. That half century resulted in a vast library of experience, little of it truly collective (new work always tends to supplant in the biotech mind-set much of what is already in the scientific literature), many claims now irrelevant, but as a body of knowledge, containing valuable concepts sometimes waiting to be rediscovered in times more favorable to bioenergy.

Each chapter contains many references to published articles (both print and electronic); these might best be viewed as akin to Web site links — each offers a potentially large amount of primary information and further links to a nexus of data and ideas. Most of the references cited were peer-reviewed, the remainder edited or with multiple authorships. No source used as a reference requires a personal subscription or purchase — Internet searches reveal many thousands more articles in trade journals and reports downloadable for a credit card payment; rather, the sources itemized can either be found in public, university, or national libraries or are available to download freely. Because the total amount of relevant informa­tion is very large, the widest possible quotation basis is required, but (as always with controversial matters) all data and information are subject to widely differing assessments and analyses.

Meanwhile, time passes, and in late 2007, oil prices approached $100/barrel, and the immediate economic momentum for biofuels shows no signs of slackening. Hard choices remain, however, in the next two decades or, with more optimistic estimates of fossil fuel longevity, sometime before the end of the twenty-first century. Perhaps, the late Douglas Adams had been more of a visionary than anyone fully appreciated when he first dreamed of interstellar transportation systems powered by equal measures of chance and improbability and of an unremarkable, nonprime, two-digit number.

3.3.1 Metabolic Routes in Bacteria for Sugar Metabolism and Ethanol Formation

Two principal routes for glucose catabolism are known to classical biochemistry in ethanologenic bacteria: the Embden-Meyerhof-Parnas (EMP) pathway of glycolysis (also present in yeasts, fungi, plants, and animals) and, in a restricted range of bac­teria (but more widely for the catabolism of gluconic acid), the Entner-Doudoroff (ED) pathway (figure 3.4).486152 The two initial steps of the ED pathway resulting in 6-phosphogluconic acid are those of the oxidative pentose phosphate pathway, but Z. mobilis is unique in operating this sequence of reactions under anaerobic condi­tions.153 The EMP and ED pathways converge at pyruvic acid; from pyruvate, a range of fermentative products can be produced, including acids such as lactate and decar — boxylated acids such as 2,3-butanediol (see figure 2.3). In S. cerevisiae and other major ethanol-producing yeasts, ethanol formation requires only two reactions from pyruvate: pyruvate decarboxylase (PDC) catalyzes the formation of acetaldehyde and ADH catalyzes the NADH-oxidizing reduction of acetaldehyde to ethanol; in E. coli, in marked contrast, no PDC naturally exists and pyruvate is catabolized by the PDH reaction under aerobic conditions or by the pyruvate formate lyase (PFL) reaction under anaerobiosis. To maintain the redox balance under fermentative con­ditions, a spectrum of products is generated from glucose in E. coli and many other enteric bacterial species in which ethanol is often a minor component (table 3.5).154

3.3.2 Genetic and Metabolic Engineering of Bacteria for Bioethanol Production

3.3.2.1 Recombinant E. coli: Lineages and Metabolic Capabilities

Attempts (beginning in the 1980s) to genetically “improve” E. coli and other bacteria for efficient ethanol production with recombinant gene technology foundered because they relied on endogenous ADH activities competing with other product pathways.4 Success soon followed when the research group at the University of Florida (Gaines­ville) combined the Z. mobilis genes for PDC and ADH with that bacterium’s homo­ethanol pathway in a single plasmid, the PET operon; in various guises, the PET system has been used to engineer ethanol production in E. coli and other bacteria.155,156 The original PET operon used the P-galactosidase promoter; to construct a vector suitable for directing chromosomal integration by transformation, a PET cartridge was devised to include the PET operon plus chloramphenicol transacetylase for selection, flanked by truncated fragments of the E. coli PFL (pfl) open reading frame (without promoter) to target homologous recombination; upon integration, PET expression was under the control of the chromosomal pfl promoter — although a single-step selection on chlor­amphenicol was required to identify a suitably highly expressing mutant.157

A crucial property of the Z. mobilis PDC is its relatively high affinity (Km) for pyruvate as a substrate: 0.4 mM as compared with 2 mM for PFL, 7 mM for lactate dehydrogenase and 0.4 mM for PDH; in consequence, much reduced amounts of lactic and acetic acids are formed, and the mix of fermentation products is much less toxic to growth and inhibitory to the establishment of dense cell populations.152 Further muta­tions have been utilized to delete genes for the biosynthesis of succinic, acetic, and lac­tic acids to further reduce the waste of sugar carbon to unwanted metabolic acids.158

When a number of the well-characterized E. coli laboratory strains were evaluated for their overall suitability for PET transformation, strain B exhibited the best hardiness to environmental stresses (ethanol tolerance and plasmid stability in nonselective media) and superior ethanol yield on xylose.159,160 This strain was isolated in the 1940s and was widely used for microbiological research in the 1960s; more importantly, it lacks all known genes for pathogenicity.161 A strain B-derived, chromosomally integrated iso­late (KO11), also with a disrupted fumarate reductase gene (for succinate formation), emerged as a leading candidate for industrial ethanol production.162 It shows ethanol production with much-improved selectivity against the mixed-acid range of fermenta­tion products[26] (figure 3.9, cf. table 3.5).157 The capabilities of KO11 have been evaluated and confirmed in laboratories in the United States and Scandinavia.43,163 An encourag­ingly wide range of carbon substrates have been shown to support ethanol production:

• Pine wood acid hydrolysates164

• Sugarcane bagasse and corn stover165

• Corn cobs, hulls, and fibers166,167

• Dilute acid hydrolysate of rice hulls168

• Sweet whey and starch169,170

• Galacturonic acid and other components in orange peel hydrolysates171

• The trisaccharide raffinose (a component of corn steep liquors and molasses)172

To increase its hardiness to ethanol and inhibitors present in acid hydrolysates of lignocel — lulosic materials, strain KO11 was adapted to progressively higher ethanol concentrations during a period of months, the culture being reselected for resistance to chlorampheni­col at regular intervals; the resulting strain (LY01) fermented 140 g/l xylose in 96 hr (compared with 120 hr with LO11).173 Increased ethanol tolerance was accompanied (fortuitously) by increased resistance to various growth inhibitors, including aromatic alcohols and acids derived from ligninolysis and various aromatic and nonaromatic alde­hydes (including 4-hydroxybenzaldehyde, syringaldehyde, vanillin, hydroxymethylfur- fural [HMF], and furfural).174-176 Of this multiplicity of inhibitors, the aromatic alcohols proved to be the least toxic to bacterial growth and metabolism, and E. coli strains can be at least as refractory to growth inhibitors as are other microbial ethanologens.

More wide-ranging genetic manipulation of ethanologenic E. coli strains has explored features of the molecular functioning of the recombinant cells as measured by quantitative gene expression and the activities of the heterologous gene prod — ucts.177-180 A long-recognized problem with high-growth-rate bacterial hosts engi­neered to contain and express multiple copies of foreign genes is that of “metabolic burden,” that is, the diversion of nutrients from biosynthesis and cell replication to supporting the expression and copying of the novel gene complement often results in a reduced growth rate in comparison with the host strain. Chromosomal integration of previously plasmid-borne genes does not avoid this metabolic demand, as became evident when attempts were made to substitute a rich laboratory growth medium with possible cheaper industrial media based on ingredients such as corn steep
liquor: uneconomically high concentrations were required to match the productivities observable in laboratory tests, but this could be only partially improved by lavish additions of vitamins, amino acids, and other putative growth-enhancing medium ingredients.177 More immediately influential was increasing PDC activity by insert­ing plasmids with stronger promoters into the chromosomally integrated KO11 strain. Because the laboratory trials in the 1990s achieved often quite low cell densi­ties (3 g dry weight of cells per liter), lower by one or two orders of magnitude than those attainable in industrial fermentations, it is highly probable that some ingenu­ity will be required to develop adequate media for large-scale fermentations while minimizing operating costs.

Genetic manipulation can, however, aid the transition of laboratory strains to commercially relevant media and the physical conditions in high-volume fermen- tors. For example, growth and productivity by the KO11 strain in suboptimal media can be greatly increased by the addition of simple additional carbon sources such as pyruvic acid and acetaldehyde; this has no practical significance because ethanol production cannot necessarily be a biotransformation from more expensive precur­sors but, together with other physiological data, implies that the engineered E. coli cells struggle to adequately partition carbon flow between the demands for growth (amino acids, etc.) and the requirement to reoxidize NADH and produce ethanol as an end product.178 179 Expressing in KO11, a B. subtilis citrate synthase, whose activ­ity is not affected by intracellular NADH concentrations, improves both growth and ethanol yield by more than 50% in a xylose-containing medium; this novel enzyme in a coliform system may act to achieve a better balance and direct more carbon to 2-oxoglutarate and thence to a family of amino acids required for protein and nucleic acid biosynthesis.178 Suppressing acetate formation from pyruvate by delet­ing the endogenous E. coli gene (ackA) for acetate kinase probably has a similar effect by altering carbon flow around the crucial junction represented by pyruvate (see figure 2.3).179

In nutrient-rich media, expressing the Z. mobilis homoethanol pathway genes in E. coli increases growth rate by up to 50% during the anaerobic fermentation of xylose.180 Gene array analysis reveals that, of the nearly 4,300 total open reading frames in the genome, only 8% were expressed at a higher level in KO11 in anaero­bic xylose fermentations when compared with the B strain parent but that nearly 50% of the 30 genes involved in xylose catabolism to pyruvate were expressed at higher levels in the recombinant (figure 3.10). Calculations from bioenergetics show that xylose is a much poorer source of biochemical energy (generating only 33% of the ATP yield per molecule oxidized), and a physiological basis for the changes in growth rate can be deduced from the genomics data in the greatly elevated expres­sions of the genes encoding the initial two enzymes of xylose catabolism, although some of the other changes in the pentose phosphate and glycolytic pathways may be important for intracellular fluxes.180

A further broadening of the substrate range of the KO11 strain was effected by expressing genes (from Klebsiella oxytoca) encoding an uptake mechanism for cello — biose, the disaccharide product of cellulose digestion; an operon was introduced on a plasmid into KO11 containing the two genes for the phosphoenolpyruvate-dependent phosphotransferase transporter for cellobiose (generating phosphorylated cellobiose)

and phospho-P-glucosidase (for hydrolyzing the cellobiose phosphate intracellu — larly).181 The K. oxytoca genes proved to be poorly expressed in the E. coli host, but spontaneous mutants with elevated specific activities for cellobiose metabolism were isolated and shown to have mutations in the plasmid that eliminated the engineered casAB promoter and operator regions; such mutants rapidly fermented cellobiose to ethanol, with an ethanol yield of more than 90% of the theoretical maximum and (with the addition of a commercial cellulase) fermented mixed-waste office paper to ethanol.

A second and distinct major lineage of recombinant E. coli was initiated at the National Center for Agricultural Utilization Research, U. S. Department of Agricul­ture, Peoria, Illinois. The starting point was the incomplete stability of the KO11 strain: phenotypic instability was reported in repeated batch or continuous cultiva­tion, resulting in declining ethanol but increasing lactic acid production.182 183 More­over, the results were different when glucose or xylose provided the carbon supply for continuous culture: on glucose alone, KO11 appeared to be stable, but ethanol productivity declined after five days, and the antibiotic (chloramphenicol) selective marker began to be lost after 30 days.184 185 Novel ethanologenic strains were created by expressing the PET operon on a plasmid in E. coli FMJ39, a strain with deleted genes for lactate dehydrogenase and PFL and, in consequence, incapable of fermen­tative growth on glucose.186 The introduced homoethanol pathway genes comple­mented the mutations and positively selected for plasmid maintenance to enable active growth by fermentation pathways, that is, self-selection under the pressure of fermenting a carbon source.187 The plasmid was accurately maintained by serial culture and transfer under anaerobic conditions with either glucose or xylose as the carbon source and with no selective antibiotic present but quickly disappeared during growth in the aerobic conditions in which the parental strain grew normally.

One of the resulting strains was further adapted for growth on xylose (FBR3); this construct can ferment a 10% (w/v) concentration of glucose, xylose, arabinose, or a mixture of all three sugars at 35°C during a period of 70-80 hr and producing up to 46.6 g/l of ethanol and at up to 91% of the theoretical maximum yield.188 The strains are also able to ferment hydrolysates prepared from corn hulls and germ meal within 60 hr and at a yield of 0.51 g ethanol/g sugar consumed.189 190 Variants of the strains have been constructed that are relatively deficient in glucose uptake because they carry a mutation in the phosphoenolpyruvate-glucose phosphotransferase system. In organisms with this transport mechanism, the presence of glucose represses the uptake of other sugars; obviating this induces the cells to utilize xylose and arabi­nose simultaneously with glucose rather than sequentially after the glucose supply begins to be exhausted and to make ethanol production more rapidly — although the overall productivity (carbon conversion efficiency) is minimally affected.191 The USDA strains will be considered further in section 3.3.2.5 when performance data for the rival bacterial ethanologens are compared.

Molecular evolution of an efficient ethanol pathway in E. coli without resorting to heterologous gene expression was finally accomplished in 2006.192 Work pub­lished over a decade earlier from the University of Sheffield had shown that in the PDH complex, the route of pyruvate oxidation in E. coli under aerobic conditions was encoded by a four-gene operon (pdhR, aceE, aceF, and Ipd), with pyruvate (or a derivative of pyruvate) acting as the inducing agent.193 A mutant of E. coli strain K12 was isolated with the essential genetic mutation occurring in the PDH operon; the phenotype was a novel pathway endowing the capacity to ferment glucose or xylose to ethanol with a yield of 82% under anaerobic conditions, combining a PDC-type enzyme activity with the endogenous ADH activity.192 This may aid the introduc­tion of bacterial ethanol production in regions (including some member states of the European Union) where genetically manipulated organisms are viewed with popular (or populist) concern.

Commercial take-up of recombinant E. coli has proved very slow, but in 2007, dem­onstration facilities using E. coli strains licensed from the University of Florida (table 3.6) have been opened or constructed in the United States and Japan (section 4.8) — in fact, the Japanese site is the world’s first commercial plant to produce ethanol from wood by a bacterial fermentation.