Many commentators state that the Oil Crisis of 1973, after the Yom Kippur War, cat­alyzed the interest in and then sustained the development of biofuels on the national and international stages. This is an overly simplistic analysis. The following words were spoken by Senator Hubert Humphrey in May 1973, some five months before war in the Middle East broke out:12

I have called these hearings because … we are concerned about what is going on with gasoline; indeed, the entire problem of energy and what is called the fuel crisis. Gas prices are already increasing sharply and, according to what we hear, they may go much higher. … We were saved from a catastrophe in the Midwest — Wisconsin, Iowa and Minnesota — and in other parts of the country, by the forces of nature and divine provi­dence. We had one of the mildest winters in the past 25 years, and had it not been for the unusually warm weather, we would have had to close schools and factories, we would have had to shut down railroads, and we would have had to limit our use of electrical power.

Security of oil supplies and the pressures of price inflation have, since the 1970s, been major issues that continue to the present day.

Even a cursory glance at figure 1.1 will show how disadvantaged were the Ger­man, Austro-Hungarian, and Ottoman empires in comparison with the Allied powers in World War I, especially after the entry of the United States in 1917, with only Polish and some Romanian oil fields beyond the vagaries of naval blockade and interception; the ingenuity of the German chemical industry was severely stretched by the effort to substitute imports (including fuel oils) by innovations with synthetic, ersatz prod­ucts. Since then, and throughout the twentieth and early twenty-first centuries, any state entering into global or regional wars faces the same strategic imperatives: how to ensure continued oil supplies and how (if possible) to control access to them. From the naval blockades of 1914 to the air strikes of the 2006 Hezbollah-Israel conflict, oil refineries and storage tanks are to be targeted, sea-lanes interdicted, and, if possible, foreign oil fields secured by invasion. In those 90 years, wars and economic depressions often demanded attempts to substitute ethanol for gasoline. In the 1920s and 1930s, several countries (Argentina, Australia, Cuba, Japan, New Zealand, the Philippines, South Africa, and Sweden) used ethanol blends in gasoline; alcohol-fueled vehicles became predominant in Germany during World War II and, by 1944, the U. S. Army had developed a nascent biomass-derived alcohol industry.11 Such programs were, however, mostly of a contingency (or emergency) nature, highly subsidized, and, once oil began flowing in increasingly large amounts after 1945, generally abandoned.

In the decade immediately preceding 1973, the United States had lost its domi­nance of world oil production (figure 1.2). Other major players were expanding (e. g., the Middle East reached 30% of world oil production) and new producers were appear­ing: Africa (Libya, Algeria, and Nigeria) already produced 13% of world oil.13 Allow­ing for inflation, world oil prices slowly decreased throughout the 1960s (figure 1.3). At the time, this was perceived as a “natural” response to increasing oil production, especially with relative newcomers such as Libya and Nigeria contributing signifi­cantly; global production after World War II followed an exponential rate of increase (figure 1.2). Political changes (especially those in Libya) and a growing cooperation between oil-producing states in the Organization of Petroleum Exporting Countries (OPEC) and the Organization of Arab Petroleum Exporting Countries (OAPEC) led to new agreements between oil producers and oil companies being negotiated in Tehran (Iran) and Tripoli (Libya) in 1970 and 1971, which reversed the real oil price erosion.

Then, Libya and Kuwait began to significantly reduce oil output in a structured, deliberate manner. In Libya, average production was reduced from a peak of 3.6 million barrels/day before June 1970 to approximately 2.2 million barrels/day in 1972 and early 1973; the Kuwaiti government enforced a ceiling of 3 million barrels/ day in early 1972, shifting down from peak production of 3.8 million barrels/day.10 Structural imbalances in the global supply of oil had by that time become apparent because of short — and medium-term causes: [3]

• Accidental damage resulted in the prolonged closure of the pipeline carry­ing oil from Saudi Arabia to the Mediterranean.

• Supply and demand became very much more closely matched, impos­ing acute pressures on shipping and refinery kinetics; the estimated spare capacity in crude oil shrank from 7 million barrels/day in 1965 to less than 0.5 million barrels/day in early 1973.

A rapid response to the outbreak of war in October 1973 continued the politically motivated reduction in crude oil output: OAPEC proposed with immediate effect to cut back output by 5% with a further 5% each month until a settlement in accord with United Nations resolutions was effected. In addition, the Gulf States of OPEC, together with Iran, imposed unilateral price rises of up to 100%. The immediate effect on world oil prices was severe (figure 1.3). More importantly, however, the effect was

not transitory: although prices decreased from the initial peaks in 1973-1974, prices began a second wave of rapid increase in 1979 after the Iranian revolution, to reach a new maximum in 1981. From more than $50 a barrel in 1981, prices then con­founded industry analysts again, despite the subsequent conflict between Iran and Iraq, and crashed down to $20 by the late 1980s, but for over a decade real oil prices had been continuously threefold more (or greater) than those paid in 1970. Although not reaching the real prices recorded in the 1860s during the American Civil War (when industrialization was a new phenomenon for most of the world), the oil price inflation between 1973 and 1981 represented a markedly different scenario from any experienced during the twentieth century — in dollar or real terms — despite world wars and major depressions (figure 1.3).

Across the industrially developed states of the Organisation for Economic Co­operation and Development (OECD) — the United States, Japan, Germany, France, United Kingdom, Italy, and Canada — while the real price of imported crude oil had decreased between 1960 and 1973 by an average of 1%/annum, the inflation-adjusted price increased by 24.5%/annum between 1973 and 1980; the result was that the oil crisis soon developed into a deep economic crisis even in those economically and technically advanced OECD nations.14 Because gasoline prices were “buffered” by the (frequently high) taxes included in the at-pump prices in the OECD countries, gasoline prices to motorists increased by only two — to threefold between 1970 and 1980, whereas crude oil prices rose by more than eightfold; in contrast, industrial and domestic oil prices increased by approximately fivefold.14

Furthermore, viewed from the perspective of 1973, the future for oil supplies to net oil importers was highly problematic. Although known oil reserves amounted to 88 x 109 tons, more than 55% of these lay in the Middle East, and mostly in OAPEC countries (figure 1.4). In the days of the then-Cold War, the Soviet Union (USSR), Eastern Europe, and China accounted for only 16.3% of world oil production but

were net exporters of both crude oil and oil products, whereas the United States had become a net importer of both (figure 1.5). In the United States, oil represented 47% of total primary energy consumption.15 In other OECD countries, the dependence on oil was even more marked: 64% in Western Europe and 80% in Japan. The developed economies of the OECD countries responded to the oil price “shocks” of the 1970s by becoming more oil-efficient: while total OECD gross domestic product (GDP) increased by 19% between 1973 and 1980, total oil imports fell by 14%, and the oil used to produce each unit of GDP fell by 20% — to offset the reduced use of oil, however, coal and (especially) nuclear energy source utilization increased greatly.16 Energy conservation became a priority (“energy-demand management” measures), and technologies for the improved efficiency of energy use were much developed, advertised, and retrofitted to both domestic and industrial premises. “Fuel switch­ing” was much less obvious in the strategies adopted by OECD countries. While the substitution of gasoline for road transport by alcohol, liquefied gas, and so forth was widely advocated, by 1980, Canada was unique in having adopted a comprehensive policy (the “off oil conversion programme”) covering all aspects of oil use and pro­viding oil reduction targets as well as financial incentives.

For an “emerging” economy like Brazil’s, the economic dislocation posed by sustained oil price rises was potentially catastrophic. In November 1973, Brazil relied on imports for more than 80% of the country’s oil consumption; in the course of the following year, the total import bill rose from $6.2 billion to $12.6 billion, and the trade balance collapsed (figure 1.6). For the preceding decade, the Brazil­ian economy had enjoyed high growth rates (figure 1.6). Industrialization had pro­ceeded well, and the inflation rate had reached its lowest level since the 1950s.17 The Brazilian government opted against economic stagnation; rather, it aimed to pay

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7

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for the higher oil bills by achieving continued growth. To meet the challenges of energy costs, the Second National Development Plan (1975-1979) decreed the rapid expansion of indigenous energy infrastructure (hydroelectricity) as well as nuclear power and alcohol production as a major means of import substituting for gasoline.

In the next decades, some of these macroeconomic targets were successfully realized. Growth rates were generally positive after 1973, and historically massive positive trade balances were recorded between 1981 and 1994. The counterindicators were, however, renewed high rates of inflation (reaching >100%/annum by 1980) and a spiral of international debt to fund developmental programs that made Brazil the
third world’s largest debtor nation and resulted in a debt crisis in the early 1980s. Arguments continue concerning the perceived beneficial and detrimental effects of the costs of developmental programs on political, social, and environmental indices in Brazil.17

Cane sugar was the key substrate and input for Brazil’s national fuel alcohol program. Sucrose production from sugarcane (Saccharum sp.) in Brazil has a long history, from its days as a colony of Portugal. Brazil had become the world’s leading sugar supplier by the early seventeenth century, but sugar production was based initially on slave labor and remained (even in the twentieth century) inefficient. This, however, represented a potential for rapid growth after 1975 because large monocul­ture plantations had been long established in the coastal regions of the northeast and southeast of the country. Expansion of cultivated land was greatly encouraged for the “modern” export crops — sugarcane, cotton, rice, corn, soybeans, and wheat — at the expense of the more traditional crops, including manioc, bananas, peanuts, and cof­fee. Sugarcane cultivation increased by 143% between 1970 and 1989 when expressed as land use, but production increased by 229% as Brazil’s historically low use of fertil­izer began to be reversed.17

Brazil is also the southernmost producer of rum as an alcoholic spirit, but cachaga is the oldest and most widely consumed national spirit beverage, with a yearly produc­tion of ca. 1.3 billion liters.18 The primary fermentation for cachaga uses sugarcane juice, and large industrial plants had been established after the end of World War II; a variety of yeasts had been developed, suitable for continuous or discontinuous fer­mentations, the former reusing and recycling the yeast cells.18 Before distillation, the fermentation is (as are all traditional potable alcohol processes) allowed to become quiescent, the yeast cells settling and then being removed (along with other residual solids) by, in technologically more advanced facilities, centrifugation; batch (“pot still”) and continuous distillation are both used, and final alcohol concentrations are in the 38 to 48% range (by volume). Predating the oil crises of the 1970s and 1980s, the first moves toward using cane sugar as a substrate for industrial ethanol produc­tion independent of beverages dated from 1930, when the Sugar and Alcohol Institute (Instituto do Agucar e do Alcool) was set up; in 1931, a decree imposed the compul­sory addition of 5% ethanol to gasoline, and the blending was increased to 10% in 1936. Four decades of experience had, therefore, been garnered in Brazil before fuel substitution became a priority on the political agenda.19

The final element in Brazil’s developing strategy to produce “gasohol” was, iron­ically, petroleum itself. Brazil had produced oil at a low rate from at least 1955, but the offshore deposits discovered by the state-owned company PETROBRAS were so large that by 1998 domestic oil production equaled 69% of domestic consumption.17 Production continued to increase (figure 1.7), and by 2005, Brazil had become a sig­nificant global producer, accounting for 2.2% of world oil production, equivalent to that of the United Kingdom, considerably higher than either Malaysia or India (both 0.9%) and approaching half that of China (4.6%).20 Indigenous refining capacity also increased during the 1970s and again after 1996 (figure 1.7). The ability to produce alcohol as a fuel or (when mixed with gasoline) as a fuel additive became — if need be, at an unquantified ecological cost (chapter 5, section 5.5.3) — an ongoing feature of Brazilian economic life.

The bacterium K. oxytoca was isolated from paper and pulp mills and grows around other sources of wood; in addition to growing on hexoses and pentoses, it can uti­lize cellobiose and cellotriose but does not secrete endoglucanase.162 A University of Florida research group transformed strain M5A1 with the xylose-directing PET operon; unlike experience with E. coli, lower plasmid copy numbers gave higher ethanol productivity than with higher plasmid copy number.218 A PET transformant could produce ethanol at up to 98% of the theoretical yield and was highly suitable for lignocellulose substrates because it utilized xylose twice as fast as glucose — and twice as fast as did E. coli strain KO11.

Stabilizing the PET operon was accomplished by chromosomal integration at the site of the PFL (pfl gene); screening for mutants hyperresistant to the selectable chloramphenicol marker resulted in the P2 strain with improved fermentation kinetics capable of producing 44-45 g/l of ethanol from glucose or cellobiose (100 g/l) within 48 hr.219 Strain P2 has been demonstrated to generate ethanol from the cellulosic and lignocellulosic materials sugarcane bagasse, corn fiber, and sugarbeet pulp.167,220,221

As a candidate industrial strain for bioethanol production, P2 can utilize a wide range of low-molecular-weight substrates, including the disaccharides sucrose, malt­ose, cellobiose, and xylobiose, the trisaccharides raffinose, cellotriose, and xylotriose, and the tetrasaccharide stachyose.172,181,219,222 This relatively nonspecific diet has led to the cloning and expression of a two-gene K. oxytoca operon for xylodextrin utilization in E. coli strain KO11; the gene product of xynB is a xylosidase (which also has weak arabinosidase activity), whereas that of the adjacent gene in the K. oxytoca genome (xynT) is a membrane protein previously found in Na+/melibiose symporters[28] and related proteins functioning in transmembrane export and import.223 The enhanced recombinant E. coli could metabolize xylodextrins containing up to six xylose residues; unexpectedly, xylodextrin utilization was more rapid than by the donor K. oxytoca.

Given the refractory nature of native lignocelluloses, it is not surprising that chemical processing techniques using acids or alkalis and elevated temperatures have been essential for their use as industrial materials. Conventionally, the starting point has been feedstock material such as wood chips, sawdust, and chopped stalks and stems from herbaceous plants.19 Mechanical size reduction is unavoidable and, therefore, has both economic and energy costs unless fragmented waste or by-products (e. g., sawdust) is the starting material.20

Diverse techniques have been explored and described for the pretreatment of size-reduced biomass materials with the aim of producing substrates that can be more rapidly and efficiently hydrolyzed — by either chemical or biological (enzymic) means — to yield mixtures of fermentable sugars. Physical and thermochemical methods described in the literature are summarized in table 2.1. These approaches have in common the use of conditions and procedures to greatly increase the surface area to which aqueous reactants and/or enzymes have access, in particular, the percentage of the major cellulosic materials that are opened up to attack and thereby reduced to glucose and oligosaccharides on hydrolysis within feasible time limits in batch or continuous processes.

Milling has been little favored because the fibrous nature of lignocellulosic materials requires lengthy processing times and unacceptably high energy inputs; only compression milling has been taken to a testing scale beyond the laboratory. Nevertheless, several studies have concluded that milling can greatly increase the susceptibility to enzymic depolymerization of cellulose.21 Irradiation with gamma rays and electron beams was a research topic from the 1950s to the 1980s; fragmentation of polysaccharides and lignin was demonstrated to increase the rates of hydrolysis of

cellulose when subsequently treated with acids or enzymes, but contradictory results, differential responses when using different wood species, and high investment costs meant that no irradiation technique progressed to pilot-scale evaluation.

Both milling and irradiation give single product streams with only minor degradation of lignocellulosic polymers. Thermochemical methods, in particular those using steam explosion,[12] can result in extensive degradation of hemicelluloses.22 Potentially, therefore, a twin-product stream process can be devised by separating solid and liquid phases, the former containing the bulk of the cellulose and the l atter the pentose and hexose components of hemicelluloses, although these may be predominantly present in oligosaccharides.23 At temperatures close to 200°C, even short (10-minute) pretreatment times have major impacts on surface area and enzyme accessibility (figure 2.4). Lignin-carbohydrate bonds are disrupted, some of the l ignin is depolymerized, and much of the morphological coherence of the lignified plant cell wall is destroyed.22 In addition, aqueous extraction at elevated temperatures removes much of the inorganic salts — this is of particular importance with feedstocks such as wheat straw whose combustion (or combustion with coal, etc.) is impeded by their high salt content and the consequent corrosion problems.2425

Chemical pretreatment methods have usually implied hydrolytic techniques using acids and alkalis, although oxidizing agents have also been considered (table 2.2). The use of such chemical reactants introduces a much higher degree of polysaccharide breakdown and greater opportunities for separately utilizing the various potential substrates in lignocellulosic materials. In fact, chemical fractionation procedures for plant cell walls have often been described and have been of inestimable value in the separation and structural elucidation of the cell wall polymers in plant cells and plant organs.26 With wheat straw, for example, sequential treatments (figure 2.5) with

FIGURE 2.4 Efficacy of steaming pretreatments with birch wood. (Data from Puls et al.23)

an aqueous methanol, sodium chlorite, and alkali yield distinct extractives, lignin, cellulose, and hemicellulose fractions.27 Similarly, sequential treatments with alkali (lignin removal) and dilute acid (hemicellulose hydrolysis) to leave a highly enriched cellulosic residue have been devised for a variety of feedstocks including switchgrass, corn cob, and aspen woodchip.28 Because the usual intention is to utilize the sugars present in the polysaccharides as fermentation substrates, however, the developments for bioindustrial applications have invariably focused on faster, simpler, and more advanced engineering options, including some that have been progressed to the pilot- plant scale. Pretreatments involving acids (including SO2 steam explosion) primarily solubilize the hemicellulose component of the feedstock; the use of organic solvents and alkalis tends, on the other hand, to cosolubilize lignin and hemicelluloses. As with thermochemical methods, the product streams can be separated into liquid and solid (cellulosic) phases; if no separation is included in the process, inevitably, a complex mixture of hexoses and pentoses will be carried forward to the fermentation step.

Combinations of physical, thermochemical, and chemical pretreatments have often been advocated to maximize cellulose digestibility by subsequent chemical or

enzymic treatments; this usually involves higher capital and processing costs, and the potential economic benefits of increased substrate accessibility have seldom been assessed in detail. Table 2.3 presents a historical sequence of pilot plants in North America, Japan, New Zealand, Europe, and Scandinavia developed for the processing of lignocellulosic feedstocks to illustrate different approaches to pretreatment choices and cellulose processing; not all of these initiatives included fermentation steps producing ethanol, but every one of them was the result of intentions to generate sugar solutions suitable for subsequent fermentative treatments. Different biomass feedstocks may require different technologies for optimized upstream processing; for example, ammonia-based pretreatments (ammonia fiber explosion and ammonia — recycled percolation) are more effective with agricultural residues (including corn stover and corn straw) than with woody materials.28 Hardwoods yield higher degrees of saccharification after steam explosion than do softwoods.22 An organic base, я-butylamine, has been recommended for pretreatment of rice straw on the evidence of efficient delignification, highly enhanced cellulose hydrolysis by cellulase, the ease of recovery of the amine, and the almost complete reprecipitation of the solubi­lized lignin when the butylamine is removed by distillation.2930

Many accounts of pretreatment optimizations have been published; over a decade ago, a report commissioned by the Energy Research and Development Corpora­tion of Australia estimated that “several thousand” articles dealt with physical and chemical pretreatment methods for lignocellulosics (including by-products from the paper and pulping industries).31 There are several reasons for this sustained research effort, including the large number of lignocellulosics of potential industrial use, the multiplicity of pretreatment methods (tables 2.1 and 2.2) singly and in combinations, uncoordinated funding from national and international agencies, and the various scales, from the laboratory bench up to demonstration units with the capacity of processing two tonnes/hr of feedstocks (table 2.3). A multiauthor review in 2005 of four thermochemical methods, two pretreatments with acid, two with ammonia, and one with lime (calcium hydroxide) as an alkali — all described as “promising tech­nologies” — concluded that although all nine approaches gave positive outcomes on increasing accessible surface area and solubilizing hemicelluloses and although all but one altered lignin structure, only five could reduce the lignin content, and only two (the ammonia-based methods) decrystallized cellulose.32 Exceptions and caveats were, however, noted; for example, ammonia fiber/freeze explosion worked well with herbaceous plants and agricultural residues and moderately well with hard­woods, but poorly with softwoods.20

Detailed comparisons of different pretreatment methods in controlled, side-by­side studies of multiple technologies using single feedstocks are very rare. A collabo­ration between the National Renewable Energy Laboratory and six universities in the United States compared ammonia explosion, aqueous ammonia recycle, controlled pH, dilute acid, flow-through with compressed hot water and lime approaches to prepare corn stover for subsequent biological conversion to sugars; material balances and energy balances were estimated for the processes, and the digestibilities of the solids were assessed by a standardized cellulase procedure.33-39 With this feedstock (already a major “waste” product resulting from the corn ethanol industry), all six pretreatment options resulted in high yields of glucose from cellulose by subsequent treatment with cellulase; in addition, the use of high-pH methods offered potential

Pilot Plants Developed for the Saccharification of Lignocellulosic Biomass

b Direct microbial conversion by cellulase-secreting ethanol producer

for reducing cellulase amounts required in cellulose hydrolysis.40 Differences were, however, observed in the kinetics of sugar release that were sufficient to influence the choice of process, enzymes, and fermentative organisms. This conclusion was foreshadowed by Swedish research on steam pretreatment of fast-growing willow (Salix) with or without SO2 impregnation that showed that, while glucose yields of more than 90% and overall xylose yields of more than 80% could be obtained both with and without SO2, the most favorable pretreatment conditions for the separate yields of glucose and xylose were closest when using SO2-impregnated wood chips.41 To a large extent, therefore, all pretreatment strategies are likely to include a partial compromise because of the very different susceptibilities to hydrolytic breakdown and solubilization of cellulose and hemicelluloses; highly efficient industrial solutions will require biotechnological approaches to provide fermenting organisms capable of using both hexoses and pentoses and both monomeric and oligomeric (and possibly polymeric) carbohydrates (this is discussed in detail in chapter 3). Even for a single choice of pretreatment method, variation in the biological material (the feedstock) will inevitably occur in, for example, the water content that will either necessitate a flexible technology or extra cost outlay to standardize and micromanage the inflow of biomass material.42

The most recent development in pretreatment technologies has been the dem­onstration that microcrystalline cellulose can be readily solubilized and recovered using a class of chemicals called “ionic liquids.” These are salts that are liquids at room temperature and stable up to 300°C; their extreme nonvolatility would also have minimal environmental impact.43 With one such ionic liquid (an я-butyl-methy- limidazolium chloride), cellulose could be solubilized by comparatively short (<3-hr) treatments at 300°C, the cellulose could then be recovered by the addition of “anti­solvents” such as water, methanol, and ethanol, and the resulting cellulose was 50­fold more susceptible to enzyme-catalyzed hydrolysis as compared with untreated cellulose.42 Chinese researchers have shown that ionic liquids can successfully pre­treat materials such as wheat straw; full commercialization still requires economic synthesis routes and toxicological assessments.44,45

The widely quoted assessment of softwoods is that, as a biomass substrate, the lig- nocellulose is too highly lignified and difficult to process to yield cellulose easily digested by cellulase — in practical terms, excess enzyme is required and imposes unrealistically high costs and protracted digestion times.170 Nevertheless, the mas­sive resources of softwood trees in the Pacific Northwest of the United States, Can­ada, Scandinavia, northern Europe, and large tracts of Russia maintain softwoods as an attractive potential biomass for fuel alcohol production. Sweden has a particular stake in maximizing the efficiency of ethanol production from softwoods on account of the planned diversion of large amounts of woody biomass to direct heat and power facilities with the phasing out of nuclear generating capacity.70 Much of the published work on softwood utilization for bioethanol indeed derives from Canadian and Scan­dinavian universities and research centers.

Although softwoods are low in xylans in comparison with other biomass crops (table 1.5), their content of glucan polymers is high; the requirement for xylose­utilizing ethanologens remains a distinct priority, whereas mannose levels are high and should contribute to the pool of easily utilized hexoses. To make the potential supply of fermentable sugars fully accessible to yeasts and bacteria for fermentation, attention has been focused on steam explosive pretreatments with or without acid catalysts (SO2 or sulfuric acid) since the 1980s; pretreatment yields a mixed pentose and hexose stream with 50-80% of the total hemicellulose sugars and 10-35% of the total glucose, whereas a subsequent cellulase digestion liberates a further 30-60% of the theoretical total glucose.70 Because only a fraction of the total glucose may be recoverable by such technologies, a more elaborate design has been explored in which a first stage is run at lower temperature for hemicellulose hydrolysis, whereas a second stage is operated at a higher temperature (with a shorter or longer heating time and with the same, higher, or lower concentration of acid catalyst) to liberate glucose from cellulose.71 Such a two-stage process results in a sugar stream (before enzyme digestion) higher in glucose and hemicellulose pentoses and hexoses but with much reduced degradation of the hemicellulose sugars and no higher levels of potential growth inhibitors such as acetic acid (figure 4.5).

Two-stage pretreatment suffers from requiring more elaborate hardware and a more complex process management; in addition, attempting to dewater sulfuric acid- impregnated wood chips before steaming decreases the hemicellulose sugar yield from the first step and the glucose yield after the second, higher-temperature stage.72 Such pressing alters the wood structure and porosity, causing uneven heat and mass transfer during steaming. Partial air drying appears to be a more suitable substrate for the dual-stage acid-catalyzed steam pretreatment.

Not only is the sugar monomeric sugar yield higher with the two-stage hydroly­sis process, the cellulosic material remaining requires only 50% of the cellulase for subsequent digestion.73 This is an important consideration because steam-pretreated softwood exhibits no evident saturation with added cellulase even at extremely high enzyme loadings that still cannot ensure quantitative conversion of cellulose to sol­uble sugars: with steam-pretreated softwood material, even lavish amounts of cel- lulase can only liberate 85% of the glucan polymer at nonviably low ratios of solid material to total digestion volume, and although high temperatures (up to 52°C) and high agitation rates are helpful, enzyme inactivation is accelerated by faster mixing speeds.74 The residual lignin left after steam pretreatment probably restricts cellulase attack and degradation by forming a physical barrier restricting access and by bind­ing the enzyme nonproductively; extraction with cold dilute NaOH reduces the lignin content and greatly increases the cellulose to glucose conversion, the alkali possibly removing a fraction of the lignin with a high affinity for the cellulase protein.75

In addition to the engineering issues, the two-stage technology has two serious economic limitations:

1. Hemicellulose sugar recovery is aided after the first step by washing the slurry with water but the amount of water used is a significant cost factor for ethanol production; to balance high sugar recovery and low water usage, a continuous countercurrent screw extractor was developed by the National Renewable Energy Laboratory that could accept low liquid to insoluble sol­ids ratios.76

2. The lignin recovered after steam explosion has a low product value on account of its unsuitable physicochemical properties; organic solvent extraction of lignin produces a higher value coproduct.77

As with other biomass substrates, heat pretreatment at extremes of pH gener­ates inhibitors of microbial ethanol production and, before this, cellulase enzyme action.7879 A number of detoxification methods have been proposed, but simply adjusting the pH to 10 to precipitate low-molecular-weight sugar and lignin deg­radation products is effective.80 Six species of yeasts — including S. cerevisiae, Candida shehatae, and species found in forest underbrush in the western United States — were tested for adaptation to softwood (Douglas fir) pretreated with dilute acid, and isolates were selected and gradually “hardened” to hydrolysate toxicity for improved ethanol production.81

Aqueous ethanol pretreatment of softwoods (the lignol process) has been strongly advocated on account of its ability to yield highly digestible cellulose as well as lignin, hemicellulose, and furfural product streams — the extraction is operated at acid pH at 185-198°C, and some sugar degradation does inevitably occur.82 After process optimization, a set of conditions (180°C, 60 minutes of treatment time, 1.25% v/v sulfuric acid, and 60% v/v ethanol) yielded83

• A solids fraction containing 88% of the cellulose present in the untreated wood chips

• Glucose and oligosaccharides equivalent to 85% of the cellulosic glucose was released by cellulase treatment (48 hours) of the treated lignocellulose

• Approximately 50% of the total xylose recovered from the solubilized fraction

• More than 70% of the lignin solubilized in a form potentially suitable for industrial use in the manufacture of adhesives and biodegradable polymers

3.1 TRADITIONAL ETHANOLOGENIC MICROBES

The fundamental challenge in selecting or tailoring a microorganism to produce eth­anol from the mixture of sugars resulting from the hydrolysis of lignocellulosic feed­stocks is easily articulated: the best ethanol producers are incompetent at utilizing pentose sugars (including those that are major components of hemicelluloses, that is, D-xylose and L-arabinose), whereas species that can efficiently utilize both pentoses and hexoses are less efficient at converting sugars to ethanol, exhibit poor tolerance of high ethanol concentrations, or coproduce high concentrations of metabolites such as acetic, lactic, pyruvic, and succinic acids in amounts to compromise the efficiency of substrate conversion to ethanol.1-4

Because bioprospecting microbial species in many natural habitats around the global ecosphere has failed to uncover an ideal ethanologen for fuel ethanol or other industrial uses, considerable ingenuity has been exhibited by molecular geneticists and fermentation specialists in providing at least partial solutions for the two most popular “combinatorial biology” strategies of

• Endowing traditional yeast ethanologens with novel traits, including the ability to utilize pentoses

• “Reforming” bacterial species and nonconventional yeasts to be more effi­cient at converting both pentoses and hexoses to ethanol

A third option, that is, devising conditions for mixed cultures to function synergisti — cally with mixtures of major carbon substrates, is discussed in chapter 4 (section 4.5).

Adding to the uncertainty is the attitude of the traditionally conservative alcohol fermentation industry toward the introduction of organisms that lack the accepted historic advantages of the yeast Saccharomyces cerevisiae in being generally regarded as safe (GRAS) and, by extrapolation, capable of being sold as an ingredi­ent in animal feed once the fermentation process is completed.5 At various times in the past 40-50 years, thermotolerant yeast strains have been developed to accelerate the fermentation process at elevated temperature.6 Bioprocesses have been advocated and, to varying degrees, developed, in which polymeric carbohydrate inputs are both hydrolyzed with secreted enzymes and the resulting sugars and oligosaccharides are taken up and metabolized to ethanol by the cell population, the so-called simultane­ous saccharification and fermentation (SSF) strategy.[18] [19] [20] Because the potential advan­tages of SSF are best understood in the light of the differing fermentation hardware requirements of multistage and single-stage fermentations, consideration of SSF technologies is postponed until the next chapter (section 4.5).

As a volatile chemical compound viewed as a gasoline substitute, pure ethanol has one major drawback. Internal combustion engines burn fuels; ethanol, in comparison with the typical hydrocarbon components of refined oils, is more oxygenated, and its combustion in oxygen generates less energy compared with either a pure hydrocarbon or a typical gasoline (table 1.1). This is not mitigated by the higher density of ethanol because liquid volumes are dispensed volumetrically and higher weights in fuel tanks represent higher loads in moving vehicles; a gallon of ethanol contains, therefore, only 70% of the energy capacity of a gallon of gasoline.1121 A review of the relative merits of alternative fuels in 1996 pointed out that ethanol not only had a higher octane number (leading to higher engine efficiencies) but also generated an increased volume of combustion products (gases) per energy unit burned; these factors in optimized ethanol engines significantly eroded the differential advantages of gasoline.21 Similar arguments could not be extended to a comparison between ethanol and diesel fuel, and ethanol had only 58 to 59% of the energy (net heat of combustion) of the latter.21

The high miscibility of ethanol and refined oil products allows a more conserva­tive option, that is, the use of low-ethanol additions to standard gasoline (e. g., E10: 90% gasoline, 10% ethanol) and requires no modifications to standard gasoline­burning vehicles. Dedicated ethanol-fueled cars were, however, the initial favorite of the Brazilian Alcohol Program (PROALCOOL); sales of alcohol-powered vehicles reached 96% of total sales in 1980 and more than 4 million such vehicles were esti­mated to be in the alcohol “fleet” by 1989.22 Such high market penetration was not, however, maintained, and sales of alcohol-powered vehicles had almost ceased by 1996 (figure 1.8). The major reason for this reversal of fortune for ethanol-fueled

vehicles was the collapse in oil prices during the late 1980s and 1990s — by 1998, the real price of crude oil was very similar to that before November 1973 (figure 1.3). Ethanol production from sugarcane in Brazil increased from a low and declining production level in early 1972, by nearly 20-fold by 1986, and then continued to increase (although at a greatly reduced rate) until 1998 (figure 1.9). The government responded to the novel “crisis” of the competing ethanol-gasoline market in several ways23: [4]

• Prices of sugarcane and ethanol were deregulated as of January 1, 1997.

• Tariffs on sugar exports were abolished in 1997.

• In January 2006, the tax rate for gasoline was set to be 58% higher than that for hydrated ethanol (93% ethanol, 7% water), and tax rates were made advantageous for any blend of gasoline and anhydrous ethanol with ethanol contents of more than 13%.

Brazilian automobile producers introduced truly flexible-fuel vehicles (FFVs) in 2003, with engines capable of being powered by gasoline, 93% aqueous ethanol, or by a blend of gasoline and anhydrous ethanol.24 In 2004, “flex-fuel” cars sold in Brazil were 16% of the total market, but during 2005, sales of FFVs overtook those of conventional gasoline vehicles (figure 1.10). This was a very “prescient” develop­ment as crude oil prices, which had been only slowly increasing during 2003 and early 2004, surged to new dollar highs in 2005 (figure 1.3). Domestic demand for ethanol-containing fuels became so great that the ethanol percentage was reduced from 25% to 20% in March 2006; this occurred despite the increased production of anhydrous ethanol for blending.25 Brazil had evolved a competitive, consumer — led dual-fuel economy where motorists made rational choices based on the relative prices of gasoline, ethanol, and blends; astute consumers have been observed to buy ethanol only when the pump price is 30% below gasoline blends — equal volumes of ethanol and gasoline are still, as noted above, divergent on their total energy (and, therefore, mileage) equivalents.

Other pertinent statistics collected for Brazil for 2004-2006 are the following:23 [5]

• Real prices for ethanol in Brazil decreased by two-thirds between 1973 and 2006.

• Sao Paolo state became the dominant contributor to national ethanol production and PETROBRAS began the construction of a 1,000-mile pipe­line from the rural interior of the state to the coast for export purposes.

A significant contraindicator is that ethanol-compatible vehicles still remain a minority of the total on Brazilian roads: in 1997, before FFVs became available, ethanol — compatible vehicles were only 21% of the total of ca. 15 million.22 The introduction of FFVs in 2005 is expected to gradually improve this ratio (figure 1.10).

Another predictable but little emphasized problem is that improvements to sugarcane harvesting methods have lead to the unemployment of 8% of seasonal sugarcane workers.23 Since 1998, Brazil has restricted the traditional practice of burning sugarcane crops (to eliminate the leaves) before manual harvesting in favor of the mechanical harvesting of green canes.19 Although far from straight­forward (because the lack of burning requires changes in pest management), this change in agricultural practice has contributed to a growing surplus of energy from sugar/alcohol plants as electricity generated on-site and offered to the distribution grids.19,22

Any overall cost-benefit of Brazil’s 30-year experience of ethanol as a biofuel is inevitably colored by the exact time point at which such an assessment is made. In April 2006, crude oil prices exceeded $70, and this price was exceeded during the summer of 2006, with crude trading briefly at $78/barrel (figure 1.11). Although the emphasis on oil prices may be perceived as one-dimensional,26 it undeniably focuses attention on real historic events, especially those on a short time scale that may, if not counterbalanced by government action and/or fiscal policies, determine the success of embryonic attempts at oil/gasoline substitution — as evidenced (negatively) by the 1990s in Brazil (figure 1.8). A survey published in 2005 by

Brazilian authors summarized many official statistics and Portuguese-language publications; the major impact factors claimed for fuel ethanol production in Brazil were the following:27

• After 1975, fuel ethanol substituted for 240 billion liters of gasoline, equivalent to $56 billion in direct importation costs and $94 billion if costs of international debt servicing are included — after 2004, the severe increases in oil prices clearly acted to augment the benefits of oil substitution (figure 1.11).

• The sugar/ethanol sector presented 3.5% of the gross national product and had a gross turnover of $12 billion, employed (directly and indirectly)

3.6 million people, and contributed $1.5 billion in taxation revenues; approximately half of the total sugarcane grown in Brazil in 2003 was dedicated to ethanol production.

• In 2004, sugarcane production required 5.6 million hectares and represented only 8.6% of the total harvested land, but more than 120 million of low — productivity pasture, natural pastures, and low-density savannas could be dedicated to sugarcane production for ethanol, with a potential ethanol yield of more than 300 billion liters/year.

Ethanol became a major exported commodity from Brazil between 1998 and 2005; exports of ethanol increased by more than 17-fold, whereas sugar exports increased by less than twofold, although price volatility has been evident with both commodities (figure 1.12).25 As a report for the International Bank for Reconstruction and Development and World Bank (first published in October 2005) noted, average

wages in the sugar-ethanol sector are higher than the mean for all sectors in Brazil.28 As a source of employment, sugarcane ethanol production directly employs more than 1 million people and is far more labor-intensive than the petrochemical indus­try: 152 times more jobs are estimated to have been created than would have been the case from an equivalent amount of petroleum products.29

Despite the apparent vibrancy of ethanol production in Brazil, ethanol use amounts to only 20 to 30% of all liquid fuels sold in Brazil.27 True levels of sub­sidies remain difficult to accurately assess; for example, public loans and state — guaranteed private bank loans were estimated to have generated unpaid debts of $2.5 billion to the Banco do Brasil alone by 1997.28 The ban on diesel-powered cars has also artificially increased fuel prices because diesel prices have been generally lower than gasohol blend prices.2728 PROALCOOL had invested $11 billion before 2005 but, by that time, could claim to have saved $11 billion in oil imports.29

Viewed from the perspectives of fermentation technology and biochemical engineering, ethanol production in Brazil improved after 1975; fermentation productivity (cubic meters of ethanol per cubic meter of fermentation tank capacity volume per day) increased by 130% between 1975 and 2000.27 This was because of continuous incremental developments and innovations; no reports of radically new fermentor designs in Brazil have been published (although very large fermentors, up to 2 million liters in capacity, are used), and ethanol concentrations in batch fermentations are in the 6 to 12% (v/v) range; the control of bacterial infection of fermentations has been of paramount importance, and selection of robust wild strains of the yeast S. cerevisiae has systematized the traditional experience that wild strains frequently overgrow “laboratory” starter cultures.30 The use of flocculent yeast strains and the adoption of continuous cultivation (chapter 4, section 4.4.1) have also been technologies adopted in Brazil in response to the increased production of sugarcane ethanol.31 Technical development of downstream technologies have been made in the largest Brazilian provider of distillation plants (Dedini S/A Industrias de Base, www. dedini. com. br): conventional (bubble cap trays), sieve tray, and azeotropic distillation methods/dehydration (cyclohexane, monoethylene glycol, and molecular sieving) processes operate at more than 800 sites — up from 327 sites before 2000.31

On a longer-term basis, genomic analysis of sugarcane promises to identify plant genes for programs to improve sugar plant growth and productivity by genetic engineering.32 33

Interest in Erwinia bacteria for ethanol production dates back at least to the late 1950s; in 1971, the explanation for the unusually high ethanol production by Erwinia species was identified as a PDC/ADH pathway, decarboxylating pyruvate to acetaldehyde fol­lowed by reduction to ethanol, akin to that in Z. mobilis; ethanol is the major fermenta­tive product, accompanied by smaller amounts of lactic acid.224 Soft-rot bacteria secrete hydrolases and lyases to solubilize lignocellulosic polymers, and the PET operon was used to transform E. carotovora and E. chrysanthemi to produce ethanol from cellobi — ose, glucose, and xylose; both strains fermented cellobiose at twice the rate shown by cellobiose-utilizing yeasts.42 The genetically engineered E. chrysanthemi could ferment sugars present in beet pulp but was inferior to E. coli strain KO11 in ethanol production, generating more acetate and succinate in mixed-acid patterns of metabolism.221

Lactococcus lactis is another GRAS organism; its use in the industrial produc­tion of lactic acid is supplemented by its synthesis of the bacteriocin nisin, the only such product approved for food preservation.225 When a PDC-encoding gene from Zymobacter palmae was inserted into L. lactis via a shuttle vector, the enzyme was functionally expressed, but, although a larger amount of acetaldehyde was detected, a slightly higher conversion of glucose to ethanol was measured (although glucose was used more slowly), and less lactic acid was accumulated, no increased ethanol production could be achieved, presumably because of insufficient endogenous ADH activity.226 The same group at USDA’s National Center for Agricultural Utilization Research examined L. plantarum as an ethanologen for genetic improvement; strain TF103, with two genes for lactate dehydrogenase deleted, was transformed with a PDC gene from the Gram-positive bacterium Sarcinia ventriculi to redirect carbon flow toward ethanol production, but only slightly more ethanol was produced (at up to 6 g/l).227 Other attempts to metabolically engineer lactic acid bacteria have been similarly unsuccessful (although more ethanol is produced than by the parental strains and the conversion of glucose to ethanol is increased by nearly 2.5-fold), the bacteria remaining eponymously and predominantly lactic acid producers; although Z. mobilis pdc and adh genes in PET operons are transcribed, the enzyme activities can be very low when compared with E. coli transformants.228 229

Zb. palmae was isolated on the Japanese island of Okinawa from palm sap by sci­entists from the Kirin Brewery Company, Yokohama, Japan. A facultative anaerobe, the bacterium can ferment glucose, fructose, galactose, mannose, sucrose, maltose, melibiose, raffinose, mannitol, and sorbitol, converting maltose efficiently to ethanol with only a trace of fermentative acids.230 Its metabolic characteristics indicate potential as an ethanologen; broadening its substrate range to include xylose followed previous work with Z. mobilis, expressing E. coli genes for xylose isomerase, xylulokinase, transaldolase, and transketolase.231 The recombinant Zb. palmae completely cofer­mented a mixture of 40 g/l each of glucose and xylose simultaneously within eight hours at 95% of the theoretical yield. Introducing a Ruminococcus albus gene for P — glucosidase transformed Zb. palmae to cellobiose utilization; the heterologous enzyme was more than 50% present on the cell surface or inside the periplasm, and the recom­binant could transform 2% cellobiose to ethanol at 95% of the theoretical yield.232 The PDC enzyme of the organism is, as discussed briefly above, an interesting target for het­erologous expression in ethanologenic bacteria; it has the highest specific activity and lowest affinity for its substrate pyruvate of any bacterial PDC, and it has been expressed in E. coli to approximately 33% of the soluble protein. Codon usage for the gene is quite similar to that for E. coli genes, implying a facile recombinant expression.233

Cyanobacteria (blue-green algae) have generally lost their fermentative capabilities, now colonizing marine, brackish, and freshwater habitats where photosynthetic metab­olism predominates; of 37 strains in a German culture collection, only five accumulated fermentation products in darkness and under anaerobic conditions, and acids (glycolic, lactic, formate, and oxalate) were the major products.234 Nevertheless, expression of Z. mobilis pdc and adh genes under the control of the promoter from the operon for the CO2-fixing ribulose 1,5-bis-phosphate carboxylase in a Synechococcus strain synthesized ethanol phototrophically from CO2 with an ethanol:acetaldehyde molar ratio higher than 75:1.235 Because cyanobacteria have simple growth nutrient require­ments and use light, CO2, and inorganic elements efficiently, they represent a sys­tem for longer-term development for the bioconversion of solar energy (and CO2) by genetic transformation, strain and process evolution, and metabolic modeling. The U. S. Department of Energy (DOE) is funding (since 2006) DNA sequencing studies of six photosynthetic bacteria at Washington University in St. Louis, Missouri, and the DOE’s own sequencing facility at Walnut Creek, California, using a biodiversity of organisms from rice paddies and deep ocean sources to maximize biochemical and metabolic potential.

In contrast to (thermo)chemical pretreatments, the use of microbial degradation of lignin to increase feedstock digestibility has several advantages:19

• Energy inputs are low.

• Hardware demands are modest.

• No environmentally damaging waste products are generated.

• Hazardous chemicals and conditions are avoided.

All of these features have associated economic cost savings. Against this, the need for lengthy pretreatment times and the degradation of polysaccharides (thus reduc­ing the total fermentable substrate) have acted to keep interest in biological pre­processing of lignocellulosic materials firmly in the laboratory. A careful choice of organism (usually a wood-rotting fungus) or a mixture of suitable organisms can, however, ensure a high degree of specificity of lignin removal.4748 Extrapolations of this approach could involve either the preprocessing of in situ agricultural areas for local production facilities or the sequential use of the biomass feedstock first as a substrate for edible mushroom production before further use of the partially depleted material by enzymic hydrolysis to liberate sugars from polysaccharides.

As with corn stover, bagasse is the residue from sugarcane juice extraction and, as such, is an obligatory waste product. Development programs for bioethanol produc­tion from bagasse started in Brazil in the 1990s, and Dedini S/A Industrias de Base (www. dedini. com. br) now operates a pilot plant facility with the capacity of produc­ing 5,000 l/day of ethanol from bagasse in Sao Paolo state; up to 109 l of hydrated alcohol can be produced per tonne of wet bagasse, and this could be increased to 180 l/tonne with full utilization of hemicellulose sugars.

The commercial Brazilian process uses organic-solvent-treated bagasse. Other reports from Cuba, Denmark, Sweden, Japan, Austria, Brazil, and the United States describe alternative processes, all with some merits:

• Steam explosion — impregnation with SO2 before steam explosion gives high yields of pentose sugars with no additional formation of toxic inhibi­tors when compared with the absence of any acid catalyst84-86

• Liquid hot water pretreatment — probably the cheapest method (requiring no catalyst or chemical) and, when operated at less than 230°C, is effec­tive at solubilizing hemicellulose and lignin while leaving cellulose as an insoluble residue for further processing87,88

• Peracetic acid — alkaline pre-pretreatment followed by the use of peracetic acid gives synergistic enhancements of cellulose digestibility89,90

• Ammonia-water mixtures — vacuum-dried material from the alkaline treatment could be used for enzymatic digestion of cellulose without wash­ing or other chemical procedure34

• Dilute acid — this has not yet been fully tested for ethanol production, but as a method for preparing xylose as a substrate for xylitol production, it is capable of yielding hydrolysates with high concentrations of free xylose91,92

• Wet oxidation — alkaline wet oxidation at 195°C for 15 minutes produces solid material that is 70% cellulase; approximately 93% of the hemicellu — loses and 50% of the lignin is solubilized, and the cellulose can be enzymi — cally processed to glucose with 75% efficiency93

Three different species of yeasts have been demonstrated to ferment pentoses and/or hexoses from chemical hydrolysates of sugarcane bagasse: S. cerevisiae, C. shehatae, Pichia stipitis, and Pachysolen tannophilus.94-97 Acid hydrolysates are

best detoxified by ion exchange materials or activated charcoal; laccase and high pH precipitation methods are less effective.95 The most recent of the reports attempted lime treatment to neutralize the acid but found that the novel technique of electrodi­alysis (migration of ions through membranes under a direct electric field) removed the sulfuric acid and also the acetic acid generated during acid hydrolysis of hemicellu — loses so effectively that the reutilization of the sulfuric acid could be contemplated.[35]97 Recombinant xylose-utilizing yeast has been desensitized to hydrolysates containing increasing concentrations of phenolic compounds, furfuraldehydes, and carboxylic acids without loss of the xylose-consuming capacity and while retaining the ability to form ethanol rather than xylitol.96 Acetic acid and furfural at concentrations similar to those measured in sugarcane bagasse hydrolysates adversely affect both “laboratory” and “industrial” strains (see chapter 3, section 3.2.4) of S. cerevisiae.98 One highly practical solution is that the predominantly pentose-containing hydrolysates from bagasse pretreatments can also be used to dilute the sugarcane juice-based medium for sugar ethanol fermentation while maintaining an equivalent sugar concentration and utilizing a pentose-consuming P. stipitis to coferment the sugar mixture.99

Sugarcane is, however, not entirely without its industrial biohazards. Bagassosis, caused by airborne cells (or fragments) of Thermoactinomyces sacchari, was once very prevalent in workforces exposed to bagasse dust. In the United States and also in Japan (where outbreaks occurred in sugar refineries and lacquerware factories), the disease is thought to have been mostly eradicated during the 1970s by improved product handling and safety practices.100,101

The principal wine yeast S. cerevisiae is, in addition to its well-known desirable prop­erties (as described above), seemingly the best “platform” choice for lignocellulose — derived substrates because strains are relatively tolerant of the growth inhibitors found in the acid hydrolysates of lignocellulosic biomass.[21] Its biotechnological limitations, on the other hand, derive from its relatively narrow range of fermentable substrates:[22]

• Glucose, fructose, and sucrose are rapidly metabolized, as are galactose and mannose (constituents of plant hemicelluloses) and maltose (a disac­charide breakdown product of starch).

• The disaccharides trehalose and isomaltose are slowly utilized, as are the trisaccharides raffinose and maltotriose (another breakdown product of starch), the pentose sugar ribose, and glucuronic acid (a sugar acid in plant hemicelluloses).

• Cellobiose, lactose, xylose, rhamnose, sorbose, and maltotetraose are nonutilizable.

product of starch) and cellobiose (a degradation product of cellulose).17 This effect is only one of four important O2-related metabolic phenomena, the others being[23]

• The Pasteur effect, that is, the inhibition of sugar consumption rate by O219

• The Crabtree effect, that is, the occurrence (or continuance) of ethanol formation in the presence of O2 at high growth rate or when an excess of sugar is provided20

• The Custers effect, that is, the inhibition of fermentation by the absence of O2 — found only in a small number of yeast species capable of fermenting glucose to ethanol under fully aerobic conditions21

For efficient ethanol producers, the probable optimum combination of phenotypes is

1. Pasteur-positive, (that is, with efficient use of glucose and other readily uti — lizable sugars for growth when O2 levels are relatively high)

2. Crabtree-positive, for high rates of ethanol production when supplied with abundant fermentable sugar from as soon as possible in the fermentation

3. Custers-negative, that is, insensitive to fluctuating, sometimes very low, O2 levels

4. Kluyver-negative, for the widest range of fermentable substrates

Fermentation of Galactose, Five Disaccharides, and Two Trisaccharides by Yeasts

K: exhibits aerobic respiratory growth but no fermentation (Kluyver effect); may include delayed use (after 7 days) Source: Data from Barnett et al.17

Not all of these effects can be demonstrated with common wine yeasts (or only under special environmental or laboratory conditions), but they are all of relevance when considering the use of novel (or nonconventional) yeasts or when adapting the growth and fermentative capacities of yeast ethanologens to unstable fermentation conditions (e. g., low O2 supply, intermittent sugar inflow) for optimum ethanol production rates. Nevertheless, S. cerevisiae, with many other yeast species, faces the serious meta­bolic challenges posed by the use of mixtures of monosaccharides, disaccharides, and oligosaccharides as carbon sources (figure 3.1). Potential biochemical “bottle­necks” arise from the conflicting demands of growth, cell division, the synthesis of cellular constituents in a relatively fixed set of ratios, and the requirement to balance redox cofactors with an inconsistent supply of both sugar substrates and O2:

• Oligosaccharide hydrolysis

• Disaccharide hydrolysis and uptake

• Hexose transport into the cells

• Conversion of hexoses to glucose 6-phosphate

• The glycolytic pathway for glucose 6-phosphate catabolism

• Pyruvate dehydrogenase (PDH) and alcohol dehydrogenase (ADH)

• The tricarboxylic (Krebs) cycle for aerobic respiration and the provision of precursors for cell growth

• Mitochondrial respiration

In the concrete circumstances represented by an individual yeast species in a partic­ular nutrient medium and growing under known physical conditions, specific combi­nations of these parameters may prove crucial for limiting growth and fermentative ability. For example, during the >60 years since its discovery, various factors have been hypothesized to influence the Kluyver effect, but a straightforward product inhibition by ethanol could be the root cause. In aerobic cultures, ethanol, suppresses the utilizability of those disaccharides that cannot be fermented, the rate of their catabolism being “tuned” to the yeast culture’s respiratory capacity.22 The physi­ological basis for this preference is that Kluyver-positive yeasts lack high-capacity transporter systems for some sugars to support the high substrate transport into cells necessary for fermentative growth, whereas energy-efficient respiratory growth sim­ply does not require a high rate of sugar uptake.23,24

The function of O2 in limiting fermentative capacity is complex; in excess, it blocks fermentation in many yeasts, but a limited O2 supply enhances fermentation in other species.10,20 Detailed metabolic analyses have shown that the basic pathways of carbon metabolism in ethanologenic yeasts are highly flexible on a quantitative basis of expression, with major shifts in how pathways function to direct the “traf­fic flow” of glucose-derived metabolites into growth and oxidative or fermentative sugar catabolism.25 26 The Crabtree effect, that is, alcoholic fermentation despite aerobic conditions, can be viewed as the existing biochemical networks adapting to consume as much of the readily available sugar (a high-value carbon source for microorganisms) as possible — and always with the possibility of being able to reuse the accumulated ethanol as a carbon source when the carbohydrate supply eventually becomes depleted.22 2728

oligosaccharides MEDIUM

A

Even when glucose fermentation occurs under anaerobic or microaerobic conditions, the fermentation of xylose (and other sugars) may still require O2. For example, when xylose metabolism commences by its reduction to xylitol (catalyzed by NADPH-dependent xylose reductase), the subsequent step is carried out under the control of an NAD-dependent xylitol dehydrogenase, thus resulting in a dis­turbed redox balance of reduced and oxidized cofactors if O2 is not present, because NADH cannot then be reoxidized, and fermentation soon ceases (figure 3.2).2930

FIGURE 3.2 Metabolic pathways of D-xylose and L-arabinose utilization by bacteria and yeasts: interconnections with oxidative and nonoxidative pen­tose phosphate pathways (for clarity, sugar structures are drawn without hydroxyl groups and both H atoms and C-H bonds on the sugar backbones).

It is precisely this biochemical complexity in yeasts that makes accurate control of an ethanol fermentation difficult and that has attracted researchers to fermenta­tive bacteria where metabolic regulation is more straightforward and where the full benefits of advances in biochemical engineering hardware and software can be more readily exploited. The remarkably high growth rate attainable by S. cerevisiae at very low levels of dissolved O2 and its efficient transformation of glucose to ethanol that made it originally so attractive for alcohol production maintain, however, its A-list status in the rankings of biologically useful organisms (figure 3.3).31 Whole genome sequencing has shown that the highly desirable evolution of the modern S. cerevisiae yeast ethanologen has occurred over more than 150 million years, result­ing in a Crabtree-positive species that can readily generate respiratory-deficient, high alcohol-producing “petite” cells immune to the Pasteur effect in a readily acquired and efficient fermentative lifestyle.32,33