Biomass — as described in the preceding section — has a calorific value that can be at least partly “captured” in other, more immediately useful forms of energy. Two of the examples included in figure 2.2 were fermentations, biological processes involving microbial bioenergetics in the chemical transformations. “Fermentation” has come a long way semantically since its coining by Louis Pasteur as life without air (la vie sans l’air).8 Pasteur was referring to yeast cells and how they altered their respiratory functions when air (or oxygen) became limiting. The modern use of the term is best understood by considering how microbes oxidize (or metabolize or combust) a substrate such as glucose released by the hydrolysis of, for example, starch or cellulose (figures 1.13 and 1.23). The complete biological oxidation of glucose can be written as

+ 6O2 ^ 6CO2 + 6H2O

As written, this chemical transformation is of little biological use to a population of microbial cells (yeast, fungi, or bacteria) because it implies only the generation of heat (metabolic heat) by the cells and that all the carbon is evolved as CO2 and cannot be utilized in cell replication. In a real microbiological scenario, glucose represents a valuable organic carbon supply as well as an energy source; part of that carbon is transformed by biochemical reactions inside the cells into the materials for new cells (before cell division), and much of the free energy that is released by the energetically favorable (exothermic) oxidation of the remainder of the glucose is used to drive the energetically unfavorable (endothermic) reactions of biosynthetic pathways; only a small portion of the total energy available is “wasted” as heat by biochemical reactions having mechanisms with thermodynamic efficiencies less than 100%:

nC6H12O6 + mO2 ^ cells + pCO2 + qH2O + evolved heat (where p, q << 6n).

Such a system is, to the microbial physiologist, simply “growth.” If, however, a metabolic output takes the form of a chemical product — especially where that product is commercially desirable or useful — then the process generates cells and product, whereas the CO2 evolved is (from an industrial or bioprocess standpoint) “waste.” In large industrial fermentors (with volumes in excess of 500,000 L), the generation of “metabolic” heat by dense cultures of microbial cells necessitates the expenditure of large amounts of energy to cool the liquid mass and, in turn, stimulates considerable efforts to recycle the “waste” heat on the industrial plant.9 Microbial processes producing antibiotics, enzymes, amino acids, and other products, recombinant proteins, flavors, and pharmaceutical active ingredients are generally termed “fermentations” although large volumes of oxygen-bearing air are supplied (via heavy-duty compressors) at high rates to the vessels so that microaerobic or anaerobic conditions are deliberately prevented, large quantities of glucose (or other carbon and energy sources) are utilized, and commercial products are accumulated as rapidly as the nutritional and physical conditions allow.

The primary fermentation in ethanol production is, on the other hand, a classic fermentation, that is, one where the ability of the cells to absorb glucose and other sugars from the medium (and to complete the early steps in their metabolic oxidation) exceeds the supply of oxygen required to fully complete the oxidative reactions. Under such conditions, yeasts such as Saccharomyces cerevisiae will accumulate (mostly) ethanol, whereas other yeasts, fungi, and bacteria produce combinations of the following:10

• Alcohols (ethanol, glycerol, я-propanol, я-butanol)

• Acids (formic, acetic, lactic, propionic, butyric)

• Decarboxylated acids (acetoin, acetone, diacetyl, 2,3-butanediol)

All these products (including ethanol) are the products (or intermediates) of a cluster of closely linked metabolic pathways and most of them are formed biosyn­thetically by reduction, thus regenerating the finite pool of redox carriers — in par­ticular, nicotinamide adenine dinucleotide (NAD) — in their oxidized forms inside the cell (figure 2.3). With the exception of ethanol (a much earlier development), all of these products were also industrially produced by microbial processes that were commercialized in the twentieth century; they represent “overflow” products of metabolism, given the supply of a high-value carbon and energy source such as glucose and an imbalanced nutritional environment where only part of the available carbon and energy can be fully used in cell replication and cell division. In particular, any cell population has, as determined by its precise genetic profile, a finite maximum specific oxygen uptake rate (i. e., grams of O2 per grams of cells per hour); feeding glucose as the growth-limiting nutrient to induce specific growth rates requiring the cells to exceed this maximum specific oxygen uptake rate causes the accumulation of “overflow” metabolites such as acetic and formic acids (figure 2.3).11 Ethanol produc­tion by S. cerevisiae, however, exhibits a highly important relationship: even under aerobic conditions (where the capacity to fully metabolize, or combust, glucose is not compromised by the lack of O2), feeding glucose at an increasingly rapid rate eventually swamps the ability of the cells to both grow and respire the glucose, that is, to provide the energy required to support growth and all the required ana­bolic, biosynthetic reactions. Below this crucial rate of glucose supply (most readily demonstrated in continuous cultures where the rate of glucose entry, and therefore, growth, is determined by the experimenter without any limitation of O2 or other nutrient supply), no ethanol is formed;[11] above this threshold, ethanol is accumulated, the “Crabtree effect” (see section 3.1.1), which is so vital to the long historical use of “wine” and other yeasts for ethanol production:10

яC6H12O6 + mO2 ^ cells + pCO2 + qH2O + rC2H5OH + evolved heat

Much has been written quasi-philosophically (and from a distinctly anthropo­centric viewpoint) on how the central metabolic pathways in microbes and plants have evolved and on their various thermodynamic inefficiencies.13 The single most pertinent point is, however, very straightforward: in a natural environment, there is no selective advantage in microbes having the capability of extracting the full thermo­dynamic energy in foodstuffs; the problem is that of kinetically utilizing (or using up) any available food source as rapidly as possible, thereby extracting as much energy

glucose

Enzyme pathways for glucose metabolism in microbial fermentations (+[2H], -[2H] represent reductive and oxidative steps,

On

On

and nutritional benefit in the shortest possible time in periods of alternating “feast and famine.”14 Many microbes are adventitious feeders, being capable of adapting even to extremes of oxygen availability in aerobic and anaerobic lifestyles. It is merely a human description that overspill products such as ethanol, acetic and lactic acids, and solvents such as acetone are “useful”; in a natural ecosystem, each might be the food­stuff for successive members of microbial consortia and lower and higher plants — in that sense of the term, nature is never wasteful. Axenic cultures of microbes in shake flasks and industrial fermentors are (in the same way as fields of monoculture crops such as soybean and sugarcane) “unnatural” because they are the products of modern agricultural practice and industrial microbiology and offer the economic advantages and potential environmental drawbacks of both of those twentieth-century activities.

Once the growth rate in a fermentation of glucose by an ethanol-forming organism becomes very low, and if glucose continues to be supplied (or is present in excess), the biochemistry of the process can be written minimally:

C6H12O6 ^ 2CO2 + 2C2H5OH

This represents a fully fermentative state where no glucose is respired and no fermentation side products are formed: for every molecule of glucose consumed, two molecules of ethanol are formed; for every 180 g of glucose consumed, 92 g of ethanol are formed, or 51.1 g of ethanol are produced per 100 g of glucose utilized. Any measurable growth or side-product formation will reduce the conversion efficiency of glucose to ethanol from this theoretical maximum.

Microbial energetics and biochemistry can, therefore, predict maximum yields of ethanol and other fermentation products from the sugars available in cane juices, starch, and cellulose (figure 2.3). How closely this theoretical maximum is reached is a function of fermentation design, control, engineering, and the biochemistry of the microorganism used for ethanol production. If a lignocellulosic substrate is to be used, however, the range of biochemical pathways required increases greatly both in scope and complexity as more sugars (pentoses as well as hexoses, plus sugar acids such as glucuronic acid) are potentially made available; this introduces substantially more variables into the putative industrial process, and preceding even this, there is the major hurdle of physically and chemically/enzymically processing biomass materials to yield — on an acceptable mass and time basis — a mixture of fermentable sugar substrates.

Switchgrass, a perennial, warm season prairie grass and the leading candidate grass energy crop, could be grown in all rural areas in the continental United States. east of the Rocky Mountains, from North Dakota to Alabama, with the exception of southern Texas, southern Florida, and Maine.20 Until the mid-1990s, switchgrass was primar­ily known in scientific agricultural publications as a forage crop for livestock. Shortly after the turn of the millennium, the species had been tested as direct energy source, co-combusted with coal at 7-10% of the energy production levels.21 Simply burning switchgrass operates at 32% energy efficiency but using pellet grass in space-heating stoves can achieve 85% conversion efficiency. The environmental and agronomic advantages of switchgrass as a direct energy crop are severalfold:21

• Like all biomass crops, emissions are low in sulfur and mercury (especially when compared with coal).

• Switchgrass requires modest amounts of fertilizer for optimum growth, much lower application rates than with corn.

• Switchgrass stands are perennial, needing no recurrent soil preparation and so greatly reducing soil erosion and runoff caused by annual tillage.

• An acre of switchgrass could be the energy equivalent of 2-6 tons of coal, the high variability being associated with fertilizer application, climate variation, and others.

In hard economic terms, however, recycling alternative fuels such as municipal solid waste and used tires has been calculated to be preferable to either switchgrass or any form of biomass, independent of the scale of use in mass burn boilers.22 This analysis is clearly restricted to what can be “acquired” for recycling, and has very differ­ent likely outcomes if lower-wastage, non-Western economies and societies were to be similarly analyzed. Using the criterion of bulk burnable material resulting from biomass drying, herbaceous plants have been advocated as the best choice for flexibly harvestable materials intended for power production via steam boilers, this choice being over that of corn stover, tree seedlings such as fast-growing willow, tree trimmings, by-products of lumber production, or switchgrass.23

Upland and lowland cultivars of switchgrass differ appreciably in their biomass yield, tolerance to drought, and response to nitrogen fertilizer application; even between both upland and lowland variants, the differences were found to be suffi­ciently great to merit recommendations for specific types of growth habitat if energy cropping were to be practiced.24 In the northern prairies, nitrogen fertilizer use results in only variable and inconsistent increases in biomass production; a single annual harvest after the first frost is optimal for polymeric material but with reduced total nitrogen and ash as well as coinciding with low “infestation” by grass weed spe­cies; a mixture of switchgrass and big blue stem grass (Andropogon gerardii Vitman) has been recommended over dependence on a monoculture approach.25 Nitrogen application was also found to be of little benefit in a 50-year trial in southern Eng­land, where five varieties of switchgrass and one of panic grass (Panicum amarum A. S. Hitchin & Chase) were compared; delaying harvest until the dead-stem stage allowed more mineral nutrients to return to the soil.26

Like all grasses, switchgrass suffers — as a substrate for bioethanol — from its low polymeric sugar content but elevated contributions of low-molecular-weight material to its dry mass; the lignin component of the insoluble material is reduced when compared with other major lignocellulosic materials (table 1.5). This subop­timal chemistry has spurred attempts to discover means to produce industrially or commercially important biomaterials from switchgrass, in particular, high-value and nutritional antioxidants.27 28 Soluble phenolics are a potential industrial resource for fine chemicals and are present in the highest concentrations in the top internodes of the grass, whereas lower internodes contained greater amounts of cell wall-linked pheno- lics such as coumaric and ferulic acids.28 Steroidal sapogenins, starting points for the synthesis of pharmacologically active compounds, are possible hepatoxins for grazing animals.29 Grass fibers can also be used as raw material for biocomposites, packagings, and thermoplastics, and switchgrass could be a large-scale substrate for fermentations to biomanufacture biodegradable polyhydroxyalkanoate polymers.30 Pulps prepared from switchgrass also show promise as reinforcement components in newsprint.31

As with the Iogen process for bioethanol, dilute acid hydrolysis has been explored as a pretreatment methodology for switchgrass; in a batch reactor, the optimum condi­tions were 1.2% sulfuric acid at 180°C for 0.5 minute; subsequent cellulase digestion released 91.4% of the cellulose as glucose and cellobiose.32 Cellulose and lignin in switchgrass pretreated with dilute acid appeared not to interact when cellulase was added to degrade the insoluble polyglucan, acid-extracted lignin having little or no effect on the rate or extent of cellulose reactivity and saccharification.33 Mixing switch — grass with aqueous ammonia and heating under pressure at 120°C for 20 minutes aided the subsequent digestion with cellulase and xylanase.34 Milder conditions were, however, developed for the ammonia fiber explosion technology — 100°C for 5 min­utes — and resulted in a 93% solubilization of the polyglucan content of the grass.35

Switchgrass has also been included as a test biomass substrate in experimental studies of simultaneous saccharification and fermentation (SSF) (section 4.5).

The previous sections have itemized the various technologies that have been developed to process lignocellulosic materials to mixtures of glucose, pentose sugars, and oligosaccharides suitable for fermentation by microbes with the production of ethanol. But what commercial and economic forces are presently acting to determine (or limit) the choice of lignocellulosic materials for the first large-scale bioethanol facilities?

The most detailed description so far available for the demonstration plant con­structed by the Iogen Corporation in Canada candidly lists the possibilities for start­ing materials:146

• Straws (wheat, barley, etc.) and corn stover as the leading candidates

• Cane bagasse as a localized leading candidate for some tropical locations

• Grass “energy crops” as possible second-generation candidates

• Native forest wood but difficult to process

• Tree farms too expensive because of demands of other markets

• Bark tree waste — cellulose and hemicellulose contents too low

• Sawdust and other mill waste — too expensive because of pulp and paper market demands

• Municipal solid waste and waste paper — too expensive because of paper demand

For a start-up lignocellulosic ethanol facility, there are crucial issues of cost and availability. An industrial plant may require, for example, close to a million tons of feedstock a year, that feedstock should be (for operational stability) as uniform as possible and as free from high levels of toxic impurities and contaminations as possible. Some materials (e. g., wood bark) have compositions that are incompatible with the high yields achieved in starch — and sugar-based materials, and some soft­woods demand unattractively high inputs of cellulase.147 Any lignocellulosic mate­rial is subject to some competitive use, and this may dictate cost considerations (table 2.10). Some of these direct competitors are long-established, mature industries, whereas others have unarguably “green” credentials for recycling waste materials or in renewable energy generation in OECD and non-OECD economies.148 Agricultural waste materials have, in addition, great potential as substrates for the “solid-state” fermentative production of a wide spectrum of fine chemicals, including enzymes, biopesticides, bioinsecticides, and plant growth regulators.149150

The enormous size of the potential supply of lignocellulose is frequently asserted; for example, “Lignocellulose is the most abundant renewable natural resource and substrate available for conversion to fuels. On a worldwide basis, terrestrial plants produce 1.3 x 1010 metric tons (dry weight basis) of wood/year, which is equivalent to 7 x 109 metric tons of coal or about two-thirds of the world’s energy requirement.”69

Some of this “wood,” however, represents trees grown (or harvested) as food crops and used as direct domestic or even industrial energy resources, while most is intimately involved in the global carbon cycle and in stabilizing the CO2 balance in the global ecosystem. Much of what is calculable as available biomass may not, therefore, be commercially harvestable on a short-term basis without the large — scale planting of dedicated “energy crops” such as fast-growing willow trees that are presently planted and harvested in Sweden for burning in district, local, and domestic heating systems.

TABLE 2.10

Competing Uses for Lignocellulosic Biomass Materials Considered for Bioethanol Production

Material Source Uses

Indeed, these were important issues addressed in a multiauthor projection of biomass options for 2030 to replace 30% of U. S. fuel demands.151 Ignoring questions of cost and efficiency of bioconversion, this study identified both forestry and agricultural resources for use in biofuels production. Three types of forest resources were quantified:

1. Primary (logging residues, removal of excess biomass in timberland fuel treatments, and fuel wood extracted from forestlands)

2. Secondary (mill residues)

3. Tertiary (urban wood residues from construction/demolition and recycling)

Similarly, lignocellulosic agricultural resources were divided among

1. Primary (crop residues, perennial grasses, and perennial woody crops)

2. Secondary (food/feed processing residues)

3. Tertiary (municipal solid waste recycling)

Approximately 280 million tons (dry weight) of such resources were estimated to be available by the time of the report on an annual basis (figure 2.12).[17] Augmentation

□ Agriculture ■ Forestry

of this supply could arise from programs to thin native forests strategically so as to reduce fire hazards — in California, for example, more than 750,000 dry tonnes/year could be generated by such activities.152 Data from Sweden (with its relatively low population density and high degree of forestation) suggest that 25% of the country’s gasoline requirements could be substituted by lignocellulose-derived ethanol from existing biomass resources.153

Of the two major immediately available sources of lignocellulosic material, however, field crop residues have the distinct advantage of being generated in close proximity to cereal crops intended (partly or entirely) for ethanol produc­tion. In August 2005, Abengoa Bioenergy (www. abengoa. com) began constructing the world’s first industrial-scale cellulosic bioethanol plant (to use wheat straw as the feedstock) immediately adjacent to its existing 195 million liters/year, Cereal Ethanol Plant (Biocarburantes de Castilla y Leon, BcyL), at Babilfuente, Salamanca, Spain, to dovetail supply trains and technologies. The biomass plant will process more than 25,000 tonnes of wheat straw and other materials to produce 5 million liters of ethanol annually in addition to preparing lignin, pentose sugars, and animal feed products as manufacturing outputs.

As an “energy crop” feedstock for bioethanol production in North America, a grass such as switchgrass (Panicum virgatum) has persuasive advantages—economic, social, and agricultural.154 But the technology for harvesting and processing grasses must be scaled up considerably from that used in, for example, silage fermentation. In the absence of such purpose-dedicated crops, intelligent choices will be mandatory to access sufficiently large supplies of suitable cellulosic feedstocks for start-up facilities.

How much biomass can any nation or region abstract without harming the environment? In a European context, this is an urgent question because any expansion of biomass use for industrial purposes brings in the threat of placing additional pressures on soil and water resources, farmland and forestry biodiversity, and may run counter to extant legislation aiming to encourage environmentally sound farming practices. The European Environment Agency naturally became interested in this issue as projections for large-scale biomass harvesting for bioenergy began to be more ambitious.155 Taking a cautious view of “harming the environment,” that is, with protected forest areas being maintained, residue removal excluded, with no grasslands or olive groves transformed into arable land, and with at least 20% of arable land maintained under environmentally friendly cultivation, the EEA estimated that by 2030 large amounts of biomass could be made available for ambitious bioenergy programs, reaching approximately 300 million tonnes of oil equivalent annually, or 15% of the total primary energy requirements. The calculated increase during the 27 years from 2003 were assumed to be the result of improved agricultural and forestry productivity and liberalization steps leading to more cultivable land being used with higher oil prices and imposed carbon taxes encouraging this expansion. The most revealing aspect of the data presented by the EEA was, however, a ranking of different plant species planted as annual crops with bioenergy as a significant end use: only a mixture of species could ensure that no increased environmental risks were likely, and it is interesting that maize (corn) ranks highest (most potentially damaging) as a monoculture (figure 2.13). The implication was that some of the

Maize Sugar Beet Potato Rape Seed Sunflower Grasses Wheat Mustard Seed Hemp Alfalfa Linseed

favored biofuel crops (maize, sugarbeet, and rapeseed) were in the highest risk category, and careful monitoring of the areas planted with those crops would be advised to minimize environmental damage; grasses were middle ranking purely on the grounds of increased fire risk.

Contamination was a consideration in the poor take-up of continuous fermentation technologies by potable alcohol producers, especially because holding prepared wort for sometimes lengthy periods without yeast inoculation provided an excellent growth medium for adventitious microbes in the brewery.177 With the accumulation of operating experience in fuel alcohol facilities, bacterial populations have been identified that not only reduce yield but also can prove difficult to eradicate; some bacteria (including lactic acid producers) form biofilms under laboratory conditions and can colonize many (perhaps every) available surface in complex sequences of linked fermentors and the associated pipe work.190

TABLE 4.2

Immobilized and Free Cell Systems for Fuel Ethanol Production: Critical Parameters for Process Efficiency

When bacterial contaminants reach 106-107 cells/ml, the economic losses for ethanol production can reach 3% of volumetric capacity; if profitability is marginal, this will have a serious impact, and antibiotic regimes have been devised to pulse controlling agents through continuous processes.191 This prophylactic approach has been applied to continuous ethanol facilities where the total losses will be greater because continuous operations have begun to dominate the larger (>40 million gal — lons/year) production plants — an antibiotic such as penicillin G is not metabolized and degraded by S. cerevisiae, and its addition rate can be poised against its expected chemical degradation at the low pH of the fermentation broth.[40] Outside the spectrum of known antibiotics, a useful alternative is the curious (and little known) chemical adjunct between urea and hydrogen peroxide; this bacteriocidal agent can effectively control lactobacilli in wheat mash and provides useful levels of readily assimilable nitrogen and O2 (by enzyme-catalyzed decomposition of the peroxide) to enhance yeast growth and fermentative capacity.192

1.1 ETHANOL FROM NEOLITHIC TIMES

There is nothing new about biotechnology. Stated more rigorously, the practical use — if not the formal or intuitive understanding of microbiology — has a very long history, in particular with regard to the production of ethanol (ethyl alcohol). The development of molecular archaeology, that is, the chemical analysis of residues on pottery shards and other artifacts recovered from archaeological strata, has begun to specify discrete chemical compounds as markers for early agricultural, horticultural, and biotechnological activities.1 Among the remarkable findings of molecular archae­ology, put into strict historical context by radiocarbon dating and dendrochronology techniques, as well as archaeobotanical and archaeological approaches, are that

• In western Asia, wine making can be dated as early as 5400-5000 BC at a site in what is today northern Iran and, further south in Iran, at a site from 3500 to 3000 BC.1

• In Egypt, predynastic wine production began at approximately 3150 BC, and a royal wine-making industry had been established at the beginning of the Old Kingdom (2700 BC).2

• Wild or domesticated grape (Vitis vinifera L. subsp. sylvestris) can be traced back to before 3000 BC at sites across the western Mediterranean, Egypt, Armenia, and along the valleys of the Tigris and Euphrates rivers. This is similar to the modern distribution of the wild grape (used for 99% of today’s wines) from the Adriatic coast, at sites around the Black Sea and southern Caspian Sea, littoral Turkey, the Caucasus and Taurus mountains, Lebanon, and the islands of Cyprus and Crete.3

• Partial DNA sequence data identify a yeast similar to the modern Saccha — romyces cerevisiae as the biological agent used for the production of wine, beer, and bread in Ancient Egypt, ca. 3150 BC.2

The occurrence of V. vinifera in regions in or bordering on the Fertile Crescent that stretched from Egypt though the western Mediterranean and to the lower reaches of the Tigris and Euphrates is crucial to the understanding of Neolithic wine making. When ripe, grapes supply not only abundant sugar but also other nutrients (organic and inorganic) necessary for rapid microbial fermentations as well as the causative yeasts themselves — usually as “passengers” on the skins of the fruit. Simply crushing (“pressing”) grapes initiates the fermentation process, which, in unstirred vessels (i. e., in conditions that soon deplete oxygen levels), produces ethanol at 5-10% by volume (approximately, 50-100 g/l).

In China, molecular archaeological methodologies such as mass spectroscopy and Fourier transform infrared spectrometry have placed “wine” (i. e., a fermented mixture of rice, honey, and grape, as well as, possibly, other fruit) as being produced in an early Neolithic site in Henan Province from 6500 to 7000 BC.4 Geographically, China lies well outside the accepted natural range of the Eurasian V. vinifera grape but is home to many other natural types of grape. Worldwide, the earliest known examples of wine making, separated by more than 2,000 km and occurring between 7000 and 9000 years ago, were probably independent events, perhaps an example on the social scale of the “convergent evolution” well known in biological systems at the genetic level.

The epithet “earliest” is, however, likely to be limited by what physical evidence remains. Before domestication of cereals and the first permanent settlements of Homo sapiens, there was a long but unrecorded (except, perhaps, in folk memory) history of hunter-gatherer societies. Grapes have, in some botanical form or other, probably been present in temperate climates for 50 million if not 500 million years.3 It would seem entirely possible, therefore, that such nomadic “tribes” — which included shamans and/or observant protoscientists — had noted, sampled, and replicated natural fermentations but left nothing for the modern archaeologist to excavate, record, and date. The presently estimated span of wine making during the last 9000 years of human history is probably only a minimum value.

Grape wines, beers from cereals (einkorn wheat, one of the “founder plants” in the Neolithic revolution in agriculture was domesticated in southeastern Turkey, ca. 8000 BC), and alcoholic drinks made from honey, dates, and other fruits grown in the Fertile Crescent are likely to have had ethanol concentrations below 10% by volume. The concentration of the ethanol in such liquids by distillation results in a wide spectrum of potable beverages known collectively as “spirits.” The evolution of this chemical technology follows a surprisingly long timeline:56 [1]

Distillation yields “95% alcohol,” a binary azeotrope (a mixture with a constant composition) with a boiling point of 78.15°C. “Absolute” alcohol, prepared by the physical removal of the residual water, has the empirical formula C2H6O and molecular weight of 46.07; it is a clear and colorless liquid with a boiling point of 78.5°C and a density (at 20°C) of 0.789 g/mL. Absolute alcohol absorbs water vapor rapidly from the air and is entirely miscible with liquid water. As a chemical known to alchemists and medicinal chemists in Europe and Asia, it found many uses as a solvent for materials insoluble or poorly soluble in water, more recently as a topical antiseptic, and (although pharmacologically highly difficult to dose accurately) as a general anesthetic. For the explicit topic of this volume, however, its key property is its inflammability: absolute alcohol has a flash point of 13°C.7

By 1905, ethanol was emerging as the fuel of choice for automobiles among engineers and motorists,* opinion being heavily swayed by fears about oil scarcity, rising gasoline prices, and the monopolistic practices of Standard Oil.8 Henry Ford planned to use ethanol as the primary fuel for his Model T (introduced in 1908) but soon opted for the less expensive alternative of gasoline, price competition between ethanol and gasoline having proved crucial. The removal of excise duty from dena­tured ethanol (effective January 1, 1907) came too late to stimulate investment in large-scale ethanol production and develop a distribution infrastructure in what was to prove a narrow window of opportunity for fuel ethanol.8

Ford was not alone in considering a variety of possible fuels for internal com­bustion engines. Rudolf Diesel (who obtained his patent in 1893) developed the first prototypes of the high-compression, thermally efficient engine that still bears his name, with powdered coal in mind (a commodity that was both cheap and readily available in nineteenth-century Germany). Via kerosene, he later arrived at the use of crude oil fractions, the marked variability of which later caused immense practi­cal difficulties in the initial commercialization of diesel engines.9 The modern oil industry had, in effect, already begun in Titusville, Pennsylvania, in the summer of 1859, with a drilled extraction rate of 30 barrels a day, equivalent to a daily income of $600.10 By 1888, Tsarist Russia had allowed Western European entrepreneurs to open up oil fields in Baku (in modern Azerbaijan) with a productive capacity of 50,000 barrels a day. On January 10, 1901, the Spindletop well in Texas began gushing, reaching a maximum flow of 62,000 barrels a day. Immediately before the outbreak of World War I, the main oil-producing countries could achieve out­puts of more than 51 million tons/year, or 1 million barrels a day. In 1902, 20,000 vehicles drove along American roads, but this number had reached more than a million by 1912. These changes were highly welcome to oil producers, including (at least, until its forced breakup in 1911) the Standard Oil conglomerate: kerosene intended for lighting domestic homes had been a major use of oil but, from the turn of the century, electricity had increasingly become both available and preferable (or fashionable). The rapid growth in demand for gasoline was a vast new market for J. D. Rockefeller’s “lost” oil companies.

Greatly aiding the industry’s change of tack was the dominance of U. S. domestic production of oil: in 1913, the oil produced in the United States amounted to more [2]

than 60% of the worldwide total (figure 1.1). The proximity within national boundar­ies of the world’s largest production line for automobiles (in Detroit) and oil refining capacities firmly cast the die for the remainder of the twentieth century and led to the emergence of oil exploration, extraction, and processing, and the related petrochemi­cal industry as the dominant features of the interlinked global energy and industrial feedstock markets.

Nevertheless, Henry Ford continued his interest in alternative fuels, sponsoring conferences on the industrial uses of agricultural mass products (grain, soybeans, etc.) in 1935-1937; the Model A was often equipped with an adjustable carburetor designed to allow the use of gasoline, alcohol, or a mixture of the two.11

This organism has been known first as Termobacterium mobile and subsequently as Pseudomonas linderi since 1912; although it was first known in Europe as a spoiling agent in cider, its function in the making of potable beverages such as palm wines is well established in Africa, Central[27] and South America, the Middle East, South Asia, and the Pacific islands and can ferment the sugar sap of the Agave cactus to yield pulque.194 The species was described as “undoubtedly one of the most unique bacterium [sic] within the microbial world.”195 Its unusual biochemistry has already

U. S. Patents Awarded for Yeast and Bacterial Ethanol Producer Strains Capable of Utilizing Lignocellulosic Substrates

Date Title Assignee Patent Number January 11, 1983 Direct fermentation of D-xylose to ethanol by a xylose-fermenting yeast mutant Purdue Research Foundation, West Lafayette, IN 4368268 September 18, 1984 Process for manufacturing alcohol by fermentation Kyowa Hakko Kogyo Co. Ltd. 4472501 December 25, 1984 Production of ethanol by yeast using xylulose Purdue Research Foundation, West Lafayette, IN 4490468 April 16, 1985 Direct fermentation of D-xylose to ethanol by a xylose-fermenting yeast mutant Purdue Research Foundation, West Lafayette, IN 4511656 May 5, 1987 Process for enhanced fermentation of xylose to ethanol United States 4663284 June 20, 1989 Process for producing ethanol from plant biomass using the fungus Paecilomyces sp. United States 4840903 March 19, 1991 Ethanol production by Escherichia coli strains co-expressing Zymomonas PDC and ADH genes University of Florida, Gainesville, FL 5000000 December 13, 1994 Combined enzyme mediated fermentation of cellulous [sic] and xylose to ethanol… United States 5372939 May 2, 1995 Xylose isomerase gene of Thermus aquaticus Nihon Shokuhin Kako Co. Ltd., Tokyo, Japan 5411886 June 13, 1995 Ethanol production by recombinant hosts University of Florida, Gainesville, FL 5424202 October 3, 1995 Process for producing alcohol CSIR, New Delhi, India 5455163 January 9, 1996 Ethanol production in Gram-positive microbes University of Florida, Gainesville, FL 5482846 May 7, 1996 Recombinant Zymomonas for pentose fermentation Midwest Research Institute, Kansas City, MO 5514583 January 27, 1998 Pentose fermentation by recombinant Zymomonas Midwest Research Institute, Kansas City, MO 5712133 March 10, 1998 Recombinant Zymomonas for pentose fermentation Midwest Research Institute, Kansas City, MO 5726053 August 4, 1998 Recombinant yeasts for effective fermentation of glucose and xylose Purdue Research Foundation, West Lafayette, IN 5789210 December 1, 1998 Single Zymomonas mobilis strain for xylose and arabinose fermentation Midwest Research Institute, Kansas City, MO 5843760

Xylose utilization by recombinant yeasts

Ethanol production in Gram-positive microbes

Recombinant cells that highly express chromosomally integrated heterologous genes

Recombinant organisms capable of fermenting cellobiose

Stabilization of PET operon plasmids and ethanol production in bacterial strains…

Genetically modified cyanobacteria for the production of ethanol…

SHAM-insensitive terminal-oxidase gene from xylose-fermenting yeast

Pentose fermentation of normally toxic lignocellulose prehydrolysate with strain of Pichia stipitis…

Recombinant Zymomonas mobilis with improved xylose ultilization

Production of ethanol from xylose

Ethanol production in recombinant hosts

Genetically modified cyanobacteria for the production of ethanol…

High-speed, consecutive batch or continuous, low-effluent process…

Transformed microorganisms with improved properties

Xyrofin Oy, Helsinki, Finland University of Florida, Gainesville, FL University of Florida, Gainesville, FL

University of Florida Research Foundation, Inc., Gainesville, FL United States

Enol Energy, Inc., Toronto, Canada

Wisconsin Alumni Research Foundation, Madison, WI

Midwest Research Institute, Kansas City, MO

Midwest Research Institute, Kansas City, MO Xyrofin Oy, Helsinki, Finland University of Florida Research Foundation, Inc., Gainesville, FL

Enol Energy, Inc., Toronto, Canada Bio-Process Innovation, Inc., West Lafayette, IN Valtion Teknillin Tutkimuskeskus, Espoo, Finland

been described (section 3.3.1), but multiple curious features of its metabolism made it a promising target for industrial process development:

• Lacking an oxidative electron transport chain, the species is energetically grossly incompetent, that is, it can capture very little of the potential bioenergy in glucose — in other words, it is nearly ideal from the ethanol fermentation standpoint.

• What little energy production is achieved can be uncoupled from growth by an intracellular wastage (ATPase).

• It shows no Pasteur effect, seemingly oblivious to O2 levels regarding glu­cose metabolism — but acetate, acetaldehyde, and acetoin are accumulated with increasing oxygenation.

During the 1970s, biotechnological interest in Z. mobilis became intense.* A patent for its use in ethanol production from sucrose and fructose was granted in mid-1989 (table 3.6). In the same year, researchers at the University of Queensland, Brisbane, Australia, demonstrated the ability of Z. mobilis to ferment industrial substrates such as potato mash and wheat starch to ethanol, with 95-98% conversion efficiencies at ethanol concentrations up to 13.5% (v/v).196 The Australian process for producing ethanol from starch was scaled up to more than 13,000 l.39

The capability to utilize pentose sugars for ethanol production — with lignocel — lulosic substrates as the goal — was engineered into a strain recognized in 1981 as a superior ethanologen; strain CP4 (originally isolated from fermenting sugarcane juice) exhibited the most rapid rate of ethanol formation from glucose, achieved the highest concentration (>80 g/l from 200 g/l glucose), could ferment both glucose and sucrose at temperatures up to 42°C, and formed less polymeric fructose (levan) from sucrose than the other good ethanol producers. On transfer to high-glucose medium, CP4 had the shortest lag time before growth commenced and one of the shortest doubling times of the strains tested.197 Researchers at the University of Sydney then undertook a series of studies of the microbial physiology and biochemistry of the organism and upscaling fermentations from the laboratory:

• Pilot-scale (500 l)-evaluated mutant strains selected for increased ethanol tolerance while improving ethanol production from sucrose and molasses became targets for strain development.198

• To reduce malodorous H2S evolution by candidate strains, cysteine auxo — trophs were isolated from studies of sulfur-containing amino acids.198

• Technical and engineering developments greatly increased the productiv­ity of selected Z. mobilis strains (as discussed in the context of bioprocess technologies in chapter 4).198

• Direct genetic manipulation was explored to broaden the substrate range.199

• High-resolution 31P nuclear magnetic resonance (NMR) of intracellular phos­phate esters in cells fermenting glucose to ethanol showed that kinetic limita­tions could be deduced early in the ED pathway (figure 3.4), in the conversion of glucose 6-phosphate to 6-phosphonogluconate, and in the glycolytic pathway (phosphoglyceromutase), defining targets for rational genetic intervention.200

By 1993, a review on Z. mobilis could already reference 362 publications.

Strains of Z. mobilis were first engineered to catabolize xylose at the National Renewable Resources Laboratory, Golden, Colorado. Four genes for xylose utilization by E. coli were introduced into Z. mobilis strain CP4 and expressed: xylose isom- erase (xylA), xylulokinase (xylB), transketolase (tktA), and transaldolase (talB) on a plasmid under the control of strong constitutive promoters from Z. mobilis.201202 The transformant CP4 (pZB5) could grow on xylose as the carbon source with an ethanol yield of 86% of the theoretical maximum; crucially, xylose and glucose could be taken up by the cells simultaneously using a permease because no active (energy — expending), selective transport system for glucose exists in Z. mobilis; the transport “facilitator” for glucose is highly specific, and only mannose and (weakly) galactose, xylose, sucrose, and fructose appear to be taken up by this mechanism.203 Using a plasmid containing five genes from E. coli, araA (encoding L-arabinose isomerase), araB (L-ribulose kinase), araD (L-ribulose 5-phosphate-4-epimerase), plus tkta and talB, a strain (ATCC39767[pZB206]) was engineered to ferment l-arabinose and produce ethanol with a very high yield (96%) but at a slow rate, ascribed to the low affinity of the permease uptake mechanism for l-arabinose.204 A third NERL strain was ATCC39767 (identified as a good candidate for lignocellulose conversion based on the evidence of its growth in yellow poplar wood acid hydrolysates) trans­formed with a plasmid introducing genes for xylose metabolism and subsequently adapted for improved growth in the presence of hydrolysate inhibitors by serial sub­culture in progressively higher concentrations of the wood hydrolyate.202205

A strain cofermenting glucose, xylose, and arabinose was constructed by chro­mosomal integration of the genes; this strain (AX101, derived from ATCC39576) was genetically stable, fermented glucose and xylose much more rapidly than it did arabinose, but produced ethanol at a high efficiency (0.46 g/g sugar consumed) and with only minor accumulations of xylitol, lactic acid, and acetic acid.206-208 The major practical drawback for the AX101 strain is its sensitivity to acetic acid (formed in lignocellulosic hydrolysates by the breakdown of acetylated sugars); this sensitivity was demonstrated in trials of the strain with an agricultural waste (oat hulls) sub­strate pretreated by the two-stage acid process developed by the Iogen Corporation in Canada, although the bacterial ethanologen outperformed a yeast in both volumet­ric productivity and glucose to ethanol conversion.209

The University of Sydney researchers have also transformed their best candidate ethanologen with the NERL pZB5 plasmid to introduce xylose utilization; strain ZM4(pZB5) produced 62 g/l of ethanol from a medium of 65 g/l of both glucose and xylose, but its ethanol tolerance was lower than that of the Z. mobilis wild type.210 The recombinant Z. mobilis shares the energy limitation on xylose observed with E. coli.180 NMR examination of strain ZM4(pZB5) growing on glucose-xylose mixtures demonstrated low levels of nucleotide phosphate sugars inside the cells when xylose was mainly supporting metabolism; because these intracellular components are bio­synthetic precursors for cell replication, the energy limitation has a clear biochemical mechanism for growth restriction.211 In addition to the metabolic burden imposed by the plasmids, the production of unwanted by-products (xylitol, acetate, lactate, acetoin, and dihydroxyacetone) and the formation of xylitol phosphate as a possible inhibitor of enzyme-catalyzed processes may all contribute to the poorer fermenta­tion performance on xylose as a carbon source. Further NMR investigations showed that acetic acid at growth-inhibitory concentrations decreased nucleotide phosphate sugars inside the cells and caused acidification of the cytoplasm, both complex bio­chemical factors difficult to remedy by genetic manipulation.212 Taking one step back, a mutant of the ZM4 strain with greater tolerance to acetate was isolated by classical selection procedures; electroporating the pZB5 plasmid into this AcR strain resulted in a xylose-fermenting strain with demonstrably improved resistance to sodium acetate at a concentration of 12 g/l.213 Overexpressing a heterologous xylulokinase gene under the control of a native Z. mobilis promoter did not, however, increase growth or xylose metabolism on a xylose-containing medium, indicating that constraints on xylose uti­lization reside elsewhere in the catabolic pathway or in xylose uptake.214 The xylulo — kinase-catalyzed step was more convincingly rate-limiting for xylose utilization with a Z. mobilis strain constructed at the Forschungszentrum Julich (Germany) with K. pneumoniae XI and XK, as well as E. coli transketolase and transaldolase genes; overexpression of XK was deduced to be necessary and sufficient to generate strains capable of fermenting xylose to ethanol at up to 93% of the theoretical yield.215

The potential impact of the acetate inhibition of Z. mobilis is so severe with com­mercial process that investigations of the effect have continued to explore new molecu­lar targets for its abatement. With starting acetate concentrations in the range 0-8 g/l in fermentations of glucose and xylose mixtures, high acetate slowed the increase in intracellular ATP (as a measure of bioenergetic “health”).216 Expressing a gene from E. coli encoding a 24-amino acid proton-buffering peptide protects Z. mobilis strain CP4 from both low pH (<3.0) and acetic acid; optimization of this strategy may be success­ful with high-productivity strains for lignocellulose hydrolysate fermentation.217

2.3.1 Lignocellulose as a Chemical Resource

Cellulose, hemicellulose, and lignin are the polymers that provide the structural rigid­ity in higher plants that grow vertically from a few centimeters to tens of meters — the giant redwood (Sequoiadendron giganteum) reaches up to 300 ft (90 m) in height. Although a multitude of microorganisms can elaborate enzymes to degrade cellulose and hemicelluloses, the success of the lignocellulose architecture in the global eco­system is such that it was only with the advent of Homo sapiens with flint and (later) metal axes that the domination of deciduous and coniferous forests (especially in the Northern Hemisphere) was seriously challenged.

At the cellular level, plants derive their remarkable resilience to physical and microbial weathering and attack from having evolved the means to greatly thicken their cell walls, using cellulose in linear polymers of high molecular weight (500,000-1,500,000) that are overlapped and aggregated into macroscopic fibers.15 Linear strands of cellulose have a close molecular arrangement in fibrillar bun­dles that are sufficiently regular to have X-ray diffraction patterns characteristic of “crystals.” This not only augments the structural cohesion but also limits access by water-soluble components and enzymes, and native cellulose is essentially insolu­ble in water.

Hemicelluloses are diverse in both sugar components and structure, with polymeric molecular weights below 50,000.15 The heterogeneity of lignins is even greater; any estimate of molecular weight is highly dependent on the method used for extraction and solubilization, and average molecular weight distributions may be less than 10,000.16 Lignin and hemicelluloses may form chemically linked complexes that bind water-soluble hemicelluloses into a three-dimensional array, cemented together by lignin, that sheaths the cellulose microfibrils and protects them from enzymic and chemical degradation.151718

Corn stover is the above-ground plant from which the corn grain has been removed, and the constituent parts are leaves, stalk, tassel, corn cob, and shuck (the husk around the grains when in the intact cob); up to 30% by dry weight of the harvested plant is repre­sented by the collected grain. In one of the earliest technological and economic reviews of corn-based fuel alcohol production, corn stover was included for consideration—but solely as an alternative to coal as a boiler fuel for distillation. In late 1978, the report for the U. S. DOE estimated that corn stover would increase the final cost of fuel ethanol by 40/gallon as the use of corn stover as a fuel entailed costs roughly double those of local Illinois coal.36 The use of corn stover was, therefore, considered to be “justified only if the plant is located in an area where transportation cost would cause a doubling of the coal cost, or environmental considerations would rule against the use of coal; neither of which is very likely.” Such arguments left corn stover in the field as an aid against soil erosion for over a decade until the option of lignocellulosic ethanol began to be seriously considered. By 2003, the National Renewable Energy Laboratory, Golden, Colorado, estimated the annual and sustainable production of corn stover as 80-100 million dry tonnes/year, of which 20% might be utilized in the manufacture of “fiber” products and fine chemicals (e. g., furfural); 60-80 million dry tonnes would remain as a substrate for bioethanol production.37 Five years earlier, an estimate of total corn stover availability had been more than 250 million tonnes, with 30 million being left on the fields for erosion control, leaving 100 million available for biofuels production.38 With expand­ing corn acreage and a definite future for corn-based ethanol, a supply of corn stover is ensured — and commercial drivers may direct that starch ethanol and “bioethanol” facilities might be best sited adjacent to one another (see chapter 2, section 2.6).

With corn stover rising up the rankings of biomass substrates for ethanol produc­tion in the United States and elsewhere, experimental investigations of pretreatment technologies has proliferated since 2002.39-50 From this impressive corpus of practi­cal knowledge, some reinforced conclusions are apparent: [34]

temperatures (25-55°C), the enzymic digestibility of the resulting cellulose was highly influenced by both the removal of acetylated hemicellulose residues and delignification, but deacetylation was not seriously influenced by the levels of O2 or the temperature.58 Adding a water washing to ammonia-pretreated material removed lignophenolic extractives and enhanced cellulose digestibility.59 Grinding into smaller particles increased the cellulose digestibility after ammonia fiber explosion, but the chemical compositions of the different particle size classes showed major changes in the contents of xylans and low-molecular-weight compounds (figure 4.4). This could be explained by the various fractions of corn stover being differentially degraded in smaller or larger particles on grinding; for example, the cobs are rela­tively refractive to size reduction; the smaller particle sizes after AFEX treatment were more cellulase-degradable than were larger particles. Electron microscopic chemical analysis of the surface of the pretreated material provided evidence that lignin-carbohydrate complexes (chapter 2, section 2.3.2) had been disrupted.59 The high hemicellulose content of corn cobs has been exploited in a development where aqueous ethanol-pretreated material is washed and then hydrolyzed with an endox — ylanase; food-grade xylooligosaccharides can easily be purified, and the cellulosic material is readily digestible with cellulase.60 An additional advantage of corn cobs is that they can be packed at high density, thus reducing the required water inputs and giving a high concentration in the xylan product stream.

The dominance of inorganic acids for acid pretreatment of biomass substrates has only recently been challenged by the use of maleic acid, one of the strongest organic dicarboxylic acids and a potential mimic of the active sites of hydrolase enzymes with two adjacent carboxylic acid residues at their active sites.61 In com­parison with dilute sulfuric acid, maleic acid use resulted in a greatly reduced loss of xylose at high solids loadings (150-200 g dry stover/l), resulting in 95% xylose

yields, only traces of furfural, and unconditioned hydrolysates that could be used by recombinant yeast for ethanol production; 90% of the maximum glucose release could be achieved by cellulase digestion of the pretreated stover within 160 hours.

Examination of (and experiments with) the cellulase digestion of pretreated corn stover have led to other conclusions for industrial applications:

• Studies of the binding of cellobiohydrolase to pretreated corn stover identi­fied access to the cellulose in cell wall fragments and the crystallinity of the cellulose microfibrils after pretreatment to be crucial.62

• Adding small amounts of surfactant-emulsifiers during cellulase digestion of pretreated corn stover also increased the conversion of cellulose, xylan, and total polysaccharide to sugars, by acting to disrupt lignocellulose, sta­bilize the enzyme, and improve the absorption of the enzyme to the mac­roscopic substrates.63

• With steam-pretreated corn stover, near-theoretical glucose yields could be achieved by combining xylanases with cellulase to degrade residual hemi — cellulose bound to lignocellulosic components.64

• The initial rate of cellulase catalyzed hydrolysis is influenced strongly by the cellulose crystallinity whereas the extent of cellulose digestion is most influ­enced by the residual lignin.65 Modern methods of polymer analysis (e. g., diffusive reflectance infrared and fluorescence techniques) used in this work may be adaptable to on-site monitoring of pretreated biomass substrates.

• The formation of glucose from pretreated corn stover catalyzed by cellulase is subject to product inhibition, and the effects of substrate concentration and the amount (“loading”) of the enzyme are important in determining kinetic parameters.66

• Cellulase and cellobiohydrolase can both be effectively recovered from pre­treated and digested corn stover and recycled with consequent cost savings of approximately 15% (50% if a 90% enzyme recovery could be achieved).67

• The solid material used for cellulase-catalyzed hydrolysis itself is a source of potential toxic compounds produced during pretreatment but trapped in the bulk solids; activated carbon is (as discussed above) effective in removing acidic inhibitors from the liquid phase resulting from digestion of the reintroduced substrate.68

A comparative study of several methods for corn stover pretreatment concluded that alkaline methodologies had the potential to reduce the quantities of cellulase necessary in cellulose digestion but that hemicellulase activities may require supplementation.69

From the perspective of 2007, an important strategic crossroads in the means of providing bioenergy has been reached. A wide complement of technologies has been developed to process realistically available amounts of lignocellulosic materials on at least a semi-industrial scale preparatory to bioethanol production.156 A crucial component in this is the massively increased production and continued improvement of the “molecular machines” of cellulases (endoglucanases).157 Chem­ical (pyrolytic and thermal “cracking”) methodologies are being critically devel­oped for biomass substrates to generate synthesis/producer gas both as a direct energy source and as an intermediary stage for “green” chemical refineries.158 Despite these advances, simple wood-burning stoves for domestic use and furnaces for district heating projects still represent far larger energy gains than does any form of biofuel production.159

Nascent industrial facilities for lignocellulosic ethanol are focused on exploiting the supply of cereal crop waste materials — in particular, wheat straw — that together comprise only 30% of the presently available lignocellulosic biomass resources.151 Worldwide, the industry may have to adapt to a succession of different seasonally available feedstocks, each of which presents unique challenges to pretreatment processing; softwood trees are, for example, dominant contributors to vegetation in Canada, northern Europe, Russia, and Scandinavia, and have both dedicated and passionate advocates and a history of several decades of scientific research; their technoeconomic features and factors are discussed in chapter 4.

Biotechnology must, therefore, demonstrate that multiple carbon sources ( hexoses, pentoses, sugar acids, oligosaccharides) can be efficiently converted to ethanol, a proposal that flies in the face of the fermentation industry’s tradition of sim­ple (single), highly reproducible carbon inputs. Moreover, biochemical engineering solutions must be found to maximize the value extracted from the processed cel­lulose, hemicelluloses, and lignin. The former area is that of metabolic engineering, the latter that of bioprocess control; both are considered next as integral parts of evolving models and paradigms of bioethanol production.

A biological solution that bypasses the severe practical difficulties posed by growing ethanologens in concentrated solutions of potentially toxic hydrolysates of lignocel — lulosic materials is to replace physicochemical methods of biomass substrate hydro­lysis with enzymic breakdown (cellulase, hemicellulase, etc.) under milder conditions — especially if enzyme-catalyzed hydrolysis can be performed immediately before the uptake and utilization of the released sugars in a combined hydrolysis/fermenta — tion bioprocess. Extrapolating back up the process stream and considering a totally enzyme-based hydrolysis of polysaccharides, an “ideal” ethanol process has been defined to include193

• Lignin removal during pretreatment to minimize unwanted solids in the substrate

• Simultaneous conversion of cellulose and hemicellulose to soluble sugars

• Ethanol recovery during the fermentation to high concentrations

• Immobilized cells with enhanced fermentation productivity

An even closer approach to the ideal would use enzymes to degrade lignin suffi­ciently without resort to extremes of pH to fully expose cellulose and hemicellulose before their degradation to sugars by a battery of cellulases, hemicellulases, and ancillary enzymes (esterases, etc.) in a totally enzymic process with only a minimal biomass pretreatment, that is, size reduction. No such process has been devised, but because pretreatment methods could solubilize much of the hemicellulose, two dif­ferent approaches were suggested in 1978 and 1988 with either cellulolytic microbes (whole cell catalysis) or the addition of fungal cellulase and hemicellulase to the fermentation medium.194 195 These two options have become known as direct micro­bial conversion (DMC) and SSF, respectively.

DMC suffers from the biological problems of low ethanol tolerance by the (usually) clostridial ethanologens and poor ethanol selectivity of the fermentation (see section 3.3.2.5).196 Commercialization has been slow, few studies progressing beyond the laboratory stage. The phytopathogenic[41] fungus Fusarium oxysporium is the sole nonbacterial wild-type microbe actively considered for DMC; the ability of the organism to ferment xylose as well as hexose sugars to ethanol was recognized in the early 1980s, and several strains can secrete cellulose-degrading enzymes.197198 Hemicellulose sugars can also be utilized in acid hydrolysates, although with low conversion efficiencies (0.22 g ethanol per gram of sugar consumed).199 Extensive metabolic engineering of F. oxysporium is, therefore, likely to be required for an efficient ethanologen, and detailed analysis of the intracellular biochemical networks have begun to reveal potential sites for intervention.200-202

Metabolic engineering of S. cerevisiae to degrade macromolecular cellulose has been actively pursued by research groups in South Africa, the United States, Can­ada, Sweden, and Japan; fungal genes encoding various components of the cellulase complex have successfully been expressed in ethanologenic S. cerevisiae, yielding strains capable of utilizing and fermenting either cellobiose or cellulose.203-206 Cal­culations show that, based on the growth kinetics and enzyme secretion by cellulose degraders such as H. jecorina, approximately 1% of the total cell protein of a recom­binant cellulase-secreting S. cerevisiae would be required, perhaps up to 120-fold more than has been achieved to date.207,208

In contrast, SSF technologies were installed in North America in the early 1990s in production plants generating between 10 million and 64 million gallons of ethanol/year from starch feedstocks.122 In addition to starch breakdown and sugar fermentation, the technology can also include the stage of yeast propagation in a cas­caded multifermentor design (figure 4.11). Extensive research worldwide has defined some factors for successful process development:

• If yeasts are to act as the ethanologens, thermotolerant strains would per­form more in harmony with the elevated temperatures at which cellulases work efficiently.209,210

• Bacteria are more readily operated in high-temperature bioprocesses, and recombinant Klebsiella oxytoca produced ethanol more rapidly under SSF conditions than did cellobiose-utilizing yeasts; coculturing K. oxytoca and S. pastorianus, K. marxianus, or Z. mobilis resulted in increased ethanol production in both isothermal and temperature-profiled SSF to increase the cellulase activity.211

• Both K. oxytoca and Erwinia species have the innate abilities to trans­port and metabolize cellobiose, thus reducing the need to add exogenous P-glucosidase to the cellulase complex; moreover, chromosomally integrat­ing the E. chrysanthemi gene for endoglucanase and expressing the gene at a high level results in high enzyme activities sufficient to hydrolyze cellulose and even produce small amounts of ethanol in the absence of added fungal

cellulase.212,213

• Although high ethanol concentrations strongly inhibit fermentations with recombinant E. coli and glucose or xylose as the carbon substrate, SSF with cellulose and added cellulase showed a high ethanol yield, 84% of the theoretical maximum.214

• Simulations of the SSF process to identify the effects of varying the operat­ing conditions, pretreatment, and enzyme activity highlight the importance of achieving an efficient cellulose digestion and the urgent need for contin­ued R&D efforts to develop more active cellulase preparations.215

• Reducing the quantity of cellulase added to ensure efficient cellulose diges­tion would also be beneficial for the economics of the SSF concept; add­ing nonionic surfactants, polyethylene glycol, and a “sacrificial” protein to decrease nonproductive absorption of cellulase to lignin binding sites have also been demonstrated to increase cellulase action so that cellulose diges­tion efficiency can be maintained at lower enzyme:substrate ratios.8216

The importance of the quantity of cellulase added was underlined by a Swedish study that showed that reducing the enzyme loading by 50% actually increased the production cost of ethanol in SSF by 5% because a less efficient cellulose hydro­lysis reduced the ethanol yield.217 At low enzyme loading, there are considerable advantages by growing the yeast inoculum on the pretreated biomass material (bar­ley straw); the conditioned cells can be used at a reduced concentration (2 g/l, down from 5 g/l), and with an increased solids content in the SSF stage.218

The cost of commercially used fungal cellulase has decreased by over an order of magnitude because of the efforts of enzyme manufacturers after 1995.219 Multiple efforts have been made to increase the specific activity (catalytic efficiency) of cel — lulases from established and promising novel microbial sources (see section 2.4.1), and recently, the National Center for Agricultural Utilization Research, Peoria, Illi­nois, has focused on the cellulase and xylanase activities from the anaerobic fungus Orpinomyces, developing a robotic sampling and assay system to improve desirable gene mutations for enzymic activity.220-222 Inserting genes for components of the cellulase complex into efficient recombinant ethanol producers has also continued as part of a strategy to reduce the need to add exogenous enzymes; such cellulases can be secreted at levels that represent significant fractions of the total cell protein and increase ethanol production capabilities.223-227 This is of particular importance for the accumulation of high concentrations of ethanol because ethanol at more than 65 g/l inhibits the fungal (H. jecorina) cellulase commonly used in SSF studies.228

SSF has been shown to be superior to independent stages of enzymic hydrolysis and fermentation with sugarcane bagasse, utilizing more of both the cellulose and hemicelluloses.229 A continued industry-wide commitment to SSF is evident in the numbers of publications on SSF technologies applied to ethanol production with a wide variety of lignocellulosic feedstocks (table 4.3).32, 230-238 Issues of process eco­nomics are discussed in chapter 5. Prominent in the list of lignocellulosic feedstocks in table 4.3 is corn stover, a material that has the unique distinction of having a specific biocatalyst designed for its utilization.239 This fusion of the biochemical abilities of Geotrichum candidium and Phanerochaete chrysosporium points toward a long-term option for both SSF and DMC, that of harnessing the proven hypercapabilities of some known microbes to degrade lignocellulose (see section 2.4.1) and converting them to ethanologens by retroengineering into them the ethanol biochemistry of Z. mobilis (see section 3.3.2). Before then, attempts will without doubt continue to introduce fungal genes for starch degradative enzymes into candidate industrial ethanologens and explore the possible advantages from combining genetic backgrounds from two microbes into a single hybrid designed for high amylase secretion.240-242 On a paral­lel track, commercial use of food wastes such as cheese whey, a lactose-rich effluent stream, has prompted the construction of strains with P-galactosidase to hydrolyze lactose extracellularly and use both the released glucose and galactose simultaneously for ethanol production under anaerobic conditions.243

As a final option — and one that mimics the evolution of natural microbial com­munities in soils, forest leaf litter, water-logged areas, and stagnant pools — coculti­vation of a good ethanologen together with an efficient secretor of enzymes to degrade polymeric carbohydrates and/or lignocelluloses is a route avoiding introducing genetically manipulated (GM) organisms and could be adapted to continuous tech­nologies if a close control of relative growth rates and cell viabilities can be achieved. One or more of the microbial partners can be immobilized; table 4.4 includes two examples of this approach together with the cocultivation of different ethanologens to ferment glucose/xylose mixtures and pretreated lignocellulosics.244-249

Simultaneous Saccharification and Fermentation Applied to Fuel Ethanol Production from Lignocellulosic Feedstocks

TABLE 4.4

Cocultivations of Ethanologenic and Ethanologenic Plus Enzyme-Secreting Microbes for DMC/SSF Processes