The first detailed economic costing of a lignocellulosic ethanol process envisaged a process similar in outline to that eventually adopted by Iogen in Canada, where wheat straw was first pretreated with acid before fungal cellulase was used to digest the cellulose for a Saccharomyces fermentation of the liberated glucose (no pentose sugars were included as substrates at that date). Results from laboratory studies were extrapolated to a 25-million-gallon/year facility that was designed to be stand­alone and capable of generating the required fungal cellulase on site (table 5.2).

The calculated factory gate plant was much higher than that estimated for corn — derived ethanol, that is, $3.34/gallon — this cost included raw materials, utilities,

maintenance materials and labor, operating labor and supplies, the facility labora­tory, plant overhead, taxes, insurance, and depreciation but made no estimates for general and administrative, sales or research costs, profit, or any by-product credits or disposal charges. Of this total, 61% was attributed to materials, of which the wheat straw feedstock accounted for 11-12%. The other components of the price estimate and various options for reducing the total were assessed:

• Utilities and capital costs were each 14% of the total.

• Labor costs were 11% of the total.

• A high-cost peptone (proteased protein) nitrogen source was included in the fermentation media; adopting a lower-price product could reduce the cost of the product by 9%.

• The conversion of cellulose to soluble sugars was 45% (w/w basis, calcu­lated on the straw weight); increasing this to 60% could effect a 6% reduc­tion in product cost.

• Reducing the enzyme loading (ratio of enzyme to cellulose) by fourfold could reduce the product price to approximately $2/gallon.

No allowance is made in table 5.2 for capital costs or profit; on the basis of 15%/year of fixed capital for general and administrative, sales, and research costs, a 15-year life expectancy of the plant, and a 48% tax rate, the selling price for the 95% ethanol product would have to have been $4.90 or $4.50/gallon for a discounted cash flow return on investment (after taxes) or 15% or 10%, respectively.12

An independent estimate for ethanol production from corn stover, funded by the DOE for work carried out at the University of Berkeley, California, was first announced at a symposium in 1978.13 Scaling up an advanced process option with cell recycling (chapter 4, section 4.4.1) and converting the postdistillation stillage into methane by anaerobic fermentation to generate a combustible source of steam generation, a total production cost of $3.38/gallon was estimated, again with no allowance for plant profitability at a 14-million-gallon/year scale of production.

The main conclusion was, therefore, that the costs of generating fermentable sugars by enzymic hydrolysis dominated production (i. e., not-for-profit) economics; consequently, any major reduction in the cost of generating the cellulase or increase in its specific activity (or stability or ease of recovery for recycling) would be highly effective in lowering the production costs. Although federal and state initiatives in operation by 1979 offered some mitigation of the high costs of biomass-derived ethanol, the sums were small: for example, under the National Energy Act of 1978, alcohol fuels were eligible for DOE entitlements worth 50/gallon, and 16 states had reduced or eliminated entirely state gasoline taxes on gasohol mixtures, the largest amount being that in Arkansas, worth 9.50/gallon.

The fixation of gaseous molecular nitrogen, N2, to biologically utilizable nitrogen is performed as an essential part of the global nitrogen cycle by bacteria that may be either free living or exist in symbiotic associations with plants, bivalves, or marine diatoms.21 The central enzymic reduction of nitrogen to ammonia is that catalyzed by nitrogenase:

N2 + 8H+ + 8e — ^ 2NH3 + H2

Molecular hydrogen is an obligatory product of the overall reaction — which, how­ever, is so energy-expensive that most nitrogen fixers rarely evolve H2, employing another hydrogenase (“uptake hydrogenase”) to recycle the H2 via oxidation by O2 and conservation of part of the potentially released energy to support nitrogenase
action. For this reason, nitrogenase-containing microbes are not viewed as likely sources of H2 for biofuels.

Formulating cost-effective media for the recombinant microorganisms developed for broad-spectrum pentose and hexose utilization (chapter 3, sections 3.2 and 3.3) commenced in the 1990s. For pentose-utilizing E. coli, for example, the benchmark was a nutrient-rich laboratory medium suitable for the generation of high-cell-density cultures.111 Media were then assessed using the criteria that the final ethanol concen­tration should be at least 25 g/l, the xylose-to-ethanol conversion efficiency would be high (90%), and a volumetric productivity of 0.52 g/l/hr was to be attained; in a defined minimal salts medium, growth was poor, only 15% of that observed in the laboratory medium; supplementation with vitamins and amino acids improved growth but could only match approximately half of the volumetric productivity. The use of corn steep liquor as a complex nitrogen source was (as predicted from its wide industrial use in fermentations) the best compromise between the provision of a complete nutritional package with plausible cost implications for a large-scale process. As an example of the different class of compromise inherent in the use of lignocellulosic substrates, the requirement to have a carbon source with a high content of monomeric xylose and low hemicellulose polymers implied the formation of high concentrations of acetic acid as a breakdown product of acetylated sugar residues; to minimize the associated growth inhibition, one straightforward strategy was that of operating the fermentation at a relatively high pH (7.0) to reduce the uptake of the weak acid inhibitor.

In a study conducted by the National Center for Agricultural Utilization Research, Peoria, Illinois, some surprising interactions were discovered between nitrogen nutrition and ethanol production by the yeast P. stipitis.112 When the cells had ceased active growth in a chemically defined medium, they were unable to ferment either xylose or glucose to ethanol unless a nitrogen source was also provided. Ethanol pro­duction was increased by the amino acids alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, leucine, and tyrosine (although isoleucine was inhibitory); a more practical nitrogen supply for industrial fermentations consisted of a mixture of urea (up to 80% of the nitrogen) and hydrolyzed milk protein supplemented with tryptophan and cysteine (up to 60%); the use of either urea or the protein hydrolysate was less effective than the combination of both. Adding small amounts of minerals, in particular, iron, manganese, magnesium, calcium, and zinc salts as well as amino acids could more than double the final ethanol concentration to 54 g/l.

Returning to recombinant E. coli, attempts to define the minimum salts concen­tration (to avoid stress imposed by osmotically active solutes) resulted in the formu­lation of a medium with low levels of sodium and other alkali metal ions (4.5 mM) and total salts (4.2 g/l).113 Although this medium was devised during optimization of lactic acid production, it proved equally effective for ethanol production from xylose. Because many bacteria biosynthesize and accumulate internally high concentrations of osmoprotective solutes when challenged with high exogenous levels of salts, sugars, and others, modulating known osmoprotectants was tested and shown to improve the growth of E. coli in the presence of high concentrations of glucose, lactate, sodium lactate, and sodium chloride.114 The minimum inhibitory concentrations of these sol­utes was increased by either adding the well-known osmoprotectant betaine, increas­ing the synthesis of the disaccharide trehalose (a dimer of glucose), or both, and the combination of the two was more effective than either alone. Although the cells’ tol­erance to ethanol was not enhanced, the use of the combination strategy would be expected to improve growth in the presence of the high sugar concentrations that are becoming ever more frequently encountered in media for ethanol fermentations.

Accurately measuring the potential for ethanol formation represented by a cel — lulosic biomass substrate for fermentation (or fraction derived from such a material) is complex because any individual fermentable sugar (glucose, xylose, arabinose, galactose, mannose, etc.) may be present in a large array of different chemical forms: monomers, disaccharides, oligosaccharides, even residual polysaccharides. Precise chemical assays may require considerable time and analytical effort. Bioassay of the material using ethanologens in a set medium and under defined, reproducible condi­tions is preferable and more cost effective — and broadly analogous to the use of shake flask tests to assess potency of new strains and isolates and the suitability of batches of protein and other “complex” nutrients in conventional fermentation laboratories.115

5.5.1 Definitions and Semantics

Much of the public and scientific debate about biofuels has assumed that the pro­duction of bioethanol as the lead biofuel is inherently “sustainable,” mostly on the grounds that any agricultural activity is renewable, whereas the extraction of crude oil is necessarily a once-only activity, given the extremely long geological time scale of oil’s generation. As a term, “sustainability” suffers from inexactness.7879 As Patzek and Pimentel79 point out, an exact definition can be deduced from thermodynamics and is consequently defined in terms of mathematical and physical properties, that is, a cyclic process is sustainable if (and only if):

1. It is capable of being maintained indefinitely without interruption, weaken­ing, or loss of quality

2. The environment on which this process depends and into which the process expels any “waste” material is itself equally renewable and maintainable

These are very strict criteria and are not exemplified by, for example, an annual replanting of a crop plant such as maize, which depends on outlays of fossil fuel energy (for fertilizers, etc.) and which may seriously deplete the soil or minerals and contribute to soil erosion — although such a system appears to be renewed every year, that is “only” within living memory or the history of agricultural production on Earth, on a geological time scale a minuscule length of time. It is a sad fact of human agricultural activity that periodic crises have accompanied large-scale, organized farming for millennia in (among many other examples) the erosion and salt accumulation that caused the downfall of the Mesopotamian civilization, the overgrazing and poor cultivation practices that have caused the expansion of the Sahara, and the overintensive cultivation of fragile tropical rainforest soils that con­tributed to the collapse of the Mayan economy and society.80 Industrial agriculture and intensive farming are relatively recent arrivals, within the last century, and they have brought accelerated land degradation and soil erosion; agrochemical residues in water courses are a global problem, leading to eutrophication of freshwater fishing grounds and threatening the collapse of fragile ecosystems. To environmental lobby­ists, therefore, the prospect of energy crop plantations is highly unwelcome if such agronomy requires and is dependent on the heavy use of fertilizers, insecticides, and pesticides, the depletion of soil organic carbon, and (worst of all) the sequestration of limited farmland.

A more analytically useful definition of the renewability of “renewable” energy could focus on a more analyzable time scale, perhaps 160 years (approximately, the life span of the industrial activity that underpins modern society).79 Perhaps, even more appropriate would be a century, that is, most of the time in which automobiles have been fuelled by gasoline and diesel. For either option, generating a quanti­tative framework is crucial to any assessment of the practicalities of a biofuels program.

Glycerol is an obligatory coproduct of biodiesel production, glycerol forming 10% by weight of the triglycerides that act as substrates for chemical or enzymic transesteri­fication (figure 6.1). The waste stream is, however, highly impure, with the glycerol mixed with alkali, unreacted methanol, free fatty acids, and chemical degradation products. The glycerol can, at the cost of considerable expenditure of energy in gas sparging and flash distillation, be recovered at more than 99.5% purity.74 Much sim­pler is to mix the crude glycerol with methanol and substitute waste petroleum oil and heavy fuel oil as a direct fuel.75

Glycerol represents a valuable chemical resource as a potential feedstock.15 Research into value-added utilization options for biodiesel-derived glycerol has, therefore, ranged widely in the search for commercial applications using both chem­ical and biotechnological methods (table 8.3).76 For the production of 1,3-PD for polymer manufacture, glycerol as source via microbial fermentation was (5-6 years ago) more expensive than either glucose or chemical routes from ethylene oxide or acolein.3 Those economics have significantly altered now, and glycerol represents the shortest, most direct route for bioproduction of 1,3-PD, a two-reaction sequence comprising an enzyme-catalyzed dehydration followed by a reduction:

glycerol ^ 3-hydroxypropionaldehyde ^ 1,3-PD

Microbial studies have focused on the fermentative production of 1,3-propandiol by clostridial species, but this is greatly complicated by the multiplicity of other products, including и-butanol, ethanol, and acids.77 Bioproduction of a nutraceutical fatty acid derivative (marketed as a dietary supplement) by microalgae is a route to a higher-value product than bulk chemicals.78 As examples of chemical engineer­ing, recent industrial patent documents have disclosed methods for converting the biodiesel waste glycerol into dichloropropanol and an alcohol-permeable membrane to generate a purified mixture of esters, glycerol, and unreacted alcohol.7980

For many years, glycerol was not considered fermentable by E. coli but only by a limited number of related bacterial species; a landmark publication in 2006, how­ever, reported that E. coli could efficiently ferment glycerol to ethanol (and a small amount of succinic acid) provided high pH in the culture was avoided — this is a cru­cial point because growth from glycerol requires an anaplerotic step (section 8.3.1), the CO2 being generated by the pyruvate formate lyase reaction whose activity may be much reduced by high pH in the growth medium (figure 8.6).81 The application of genetically engineered E. coli with superior ethanologenic potential (chapter 3,

Chemical and Biotechnological Transformations of Biodiesel-Derived Glycerol

Fermentation route

Clostridium butyricum, Klebsiella pneumoniae None

Gluconobacter oxydans Anaerobiospirillum succiniciproducens Enterobacter aerogenes None None

Various osmophilic microbial species

section 3.3.2.1), combined with further manipulations to eliminate competing path­ways of acid accumulation, could result in ethanol productivity approaching the theoretical molar production (mole per mole) from glycerol.

Genetic Manipulation with Genes for Xylose Isomerization

Historically, the earliest attempts to engineer xylose metabolic capabilities into S. cerevisiae involved the single gene for xylose isomerase (XI, catalyzing the intercon­version of xylose and xylulose) from bacteria (E. coli and B. subtilis), but these failed because the heterologous proteins produced in the yeast cells were enzymically inac- tive.4,69 A greater degree of success was achieved using the XI gene (xylA) from the bacterium Thermus thermophilus; the transformants could exhibit ethanol formation in O2-limited xylose fermentations.95

A crucial breakthrough, however, was made in 2003, when the gene encoding XI in the fungus Piromyces sp. strain E2 (capable of anaerobic growth on xylose) was recognized as part of the known “bacterial” pathway for xylose catabolism and, for the first time, was revealed to be functional in a eukaryote.96 The same research group at the Delft Technical University, The Netherlands, soon demonstrated that xylA gene expressed in S. cerevisiae gave high XI activity but could not by itself induce ethanol production with xylose as the carbon source.97 The additional genetic manipulations required for the construction of ethanologenic strains on xylose were the overexpression of XK, transketolase, transaldolase, ribulose 5-phosphate epim — erase, and ribulose 5-phosphate isomerase and the deletion of nonspecific AR, fol­lowed by selection of “spontaneous” mutants in xylose-limited continuous cultures and anaerobic cultivation in automated sequencing-batch reactors on glucose-xylose media.98-100 The outcome was a strain with negligible accumulation of xylitol (or xylulose) and a specific ethanol production three — to fivefold higher than previously publicized strains.4 Mixtures of glucose and xylose were sequentially but completely consumed by anaerobic cultures of the engineered strain in anaerobic batch culture, with glucose still being preferred as the carbon source.99

A side-by-side comparison of XR/XDH — and XI-based xylose utilizations in two isogenic strains of S. cerevisiae with genetic modifications to improve xylose metab­olism (overexpressed XK and nonoxidative pentose phosphate pathway enzymes and deleted AR) arrived, however, at widely different conclusions for the separately opti­mal parameters of ethanol production:101

• In chemically defined medium, the Xl-containing variant showed the high­est ethanol yield (i. e., conversion efficiency) from xylose.

• The XR/XDH transformant had the higher rate of xylose consumption, spe­cific ethanol production, and final ethanol concentration, despite accumu­lating xylitol.

• In a lignocellulose hydrolysate, neither transformant accumulated xylitol, but both were severely affected by toxic impurities in the industrially rel­evant medium, producing little or no ethanol, xylitol, or glycerol and con­suming little or no xylose, glucose, or mannose.

The bacterial XI gene from T. thermophilus was revisited in 2005 when this path­way for xylose utilization was expressed in S. cerevisiae along with overexpressed XK and nonoxidative pentose phosphate pathway genes and deleted AR; the engineered strain, despite its low measured XI activity, exhibited for the first time aerobic growth on xylose as sole carbon source and anaerobic ethanol production at 30°C.102

After herbicide resistance in major crop species, the first target area for GM crop development was that of protection against plant pests.283 Although tolerance of modern herbicides is usually located in the amino acid sequences of a handful of target genes in biosynthetic pathways, plants have multiple inducible mechanisms to fight back against microbial pathogens:284 [44]

Wheat is prone to attack by the rust pathogens, Puccinia graminis and P. tritici; stem and leaf rusts are considered to be major constraints to wheat production world­wide.285 More immediately alarming from the perspective of Iogen’s dependence on wheat biomass is that new and highly infectious variants of the pathogen have been noted in Africa for some years, with newspaper reports in the first quarter of 2007 describing its spread into Asia. A single gene (Sr2) has been identified as a broad — spectrum resistance locus for more than 80 years; recently, this gene (or two tightly linked genes) confer resistance and the associated dark pigmentation traits, pseudo­black chaff.286 Stem rust-susceptible barley has been transformed into a resistant form by an Agrobacterium plasmid containing the barley resistance gene, RpgP; a single copy of the gene is sufficient to confer resistance against stem rust.287

Of the abiotic stresses that plants experience, drought is a serious limiting factor on growth and productivity even in the Northern Hemisphere, and cereal crops are highly prone to fluctuating yield depending on seasonal rainfall and average tempera­tures in the growing season; to be dependent on a monoculture crop such as wheat (for starch or straw) runs the risk of uncertain prices as well as variable feedstock availability. With the evolution of domesticated cereal species over millennia, genetic diversity has been lost; using the natural genetic diversity of wild species is an invalu­able resource because wild types harbor very broad ranges of tolerance characteris­tics; other exploitable traits include those for salt tolerance (allowing saline water to be the source of irrigation) and, especially if global temperatures increase because of global warming, flowering times and other growth parameters more typical of Medi­terranean regions for transfer to cultivars grown further north.288 Drought appears to exert physiological effects via oxidative stress signaling pathways, a property shared with freezing, heat, and salinity stresses; protein kinases are often associated with signaling pathways and expression of a protein kinase gene (NPK1) from tobacco in maize protects kernel weights when the water supply is reduced.289

6.3.1 Methanol: Thermochemical and Biological Routes

The versatility of the FT process is that almost any hydrocarbon produced that can be derived from petroleum can be made from syngas, not only alkanes and alkenes but oxygenated compounds; the exact mixture of products obtained can be varied by choices of catalyst, pressure, and temperature, and straight-chain alcohols are pro­duced in the “synthol” reaction at 400-450°C and 14-MPa pressure in the presence of an iron catalyst.94 Industrial production from natural gas has, however, been dom­inated since the 1960s by a lower temperature and pressure process invented by Imperial Chemical Industries in which CO, CO2, and H2, derived by steam reform­ing, are reacted over a mixed Cu/ZnO/Al2O3 catalyst at 250°C and 50-10 MPa when two reactions occur:95

CO + 2H2 ^ CH3OH
CO2 + 3H2 ^ CH3OH + H2O

A recent development has been to combine syngas production from methane with the reduction of ZnO to metallic zinc in a metallurgical plant; the syngas has a H2:CO ratio of approximately 2:1, highly suitable for methanol production.96 A renewable-resource route for methanol (one of the largest bulk chemicals in the contemporary world) is, however, entirely feasible via biomass gasification as an intermediate step, and this would be entirely appropriate given methanol’s older name of “wood alcohol,” indicative of its historical provenance by incomplete combustion.

As an “energy carrier,” methanol is inferior to ethanol, with an energy content of only 75% (on either a weight or a volume basis) compared with ethanol and approximately 50% compared with conventional gasoline.97 Blends of methanol with conventional gasoline up to 20% can be tolerated without the need for engine modifications, that is, as a fuel extender; the corrosive effect of methanol on some engine materials limits the extent of this substitution.98 Methanol would have been an excellent replacement for MTBE as a gasoline oxygenate additive (chapter 1, section 1.4), but its acute neurotoxicity is well known and a barrier to several poten­tial uses. One notable exception, however, is as an at-site (or on-board) source of hydrogen for fuel cells (chapter 7, section 7.1): between 1983 and 2000, nearly 50 patents were granted for catalytic methanol “reforming” systems to automobile producers (such as General Motors, Daimler-Benz, DaimlerChrysler, and Honda), chemical multinationals (DuPont, BASF, etc.), and major oil companies (CON­OCO, Standard Oil Company, etc.).95 Combined reforming with liquid water and gaseous oxygen has been intensively investigated for use in mobile applications for transportation:

(s+p)CH3OH (l) + sH2O (l) + 0.5pO2 ^ (s+p)CO2 + (3s+2p)H2

since the composition of the reactant feed can be varied and the process carried out under a wide range of operating conditions.[61]

The first pilot plant for testing and evaluating the production process for “bio­methanol” was established in a program that commenced in 2000 between the Minis­try of Agriculture, Forestry, and Fisheries of Japan and Mitsubishi Heavy Industries at Nagasaki (Japan); various feedstocks have been investigated, including wood, rice husks, rice bran, and rice straw.99 The test plant consisted of

• A drier and grinder for the biomass input (crushed waste wood)

• A syngas generator

• A gas purifier

• A methanol synthesis vessel (with an unspecified catalyst)

The pilot plant was designed for a capacity of 240 kg/day, with a methanol yield (weight of methanol produced per unit dry weight of material) of 9-13%. A larger plant (100-tonne daily capacity) is predicted to have a much higher methanol yield (38-50%).

No economic analysis of the Japanese pilot facility has been published, but a the­oretical study of methanol production via the syngas route suggested that methanol from biomass (by 2002) had production costs approximately twice those of conven­tional gasoline on an equal energy basis.100 The surge in crude oil and gasoline refin­ery gate costs subsequently (figure 5.1) implies that methanol production costs from biomass sources would now (mid-2008) be competitive. This encouraging result is very timely because direct methanol fuel cell (DMFC) technology has reached the stage where Toshiba in Japan has announced the development of a micro-DMFC suitable for powering MP3 players.101 A U. S. patent covering aspects of DMFC con­struction was also issued in March 2007 to Creare (Hanover, New Hampshire).102 The Japanese invention utilizes a polymer electrolyte membrane device with the electrochemical reactions:

CH3OH + H2O ^ CO2 + 6H+ + 6e — (anode)

1.5O2 + 6H+ + 6e — ^ 3H2O (cathode)

The inputs are concentrated methanol and air (O2); the only outputs are water and CO2 and electricity (100 mW) sufficient to power a portable device for 20 hours on a 2-cm3 charge of solvent. The prospects for large DMFCs for heavier duty use are presently unclear.

Another thermochemical route has been explored to convert methanol to another biofuel, dimethylether (DME), (CH3)2O, a highly volatile liquid that is a suitable fuel for diesel engines because of its low self-ignition temperature and high cetane num — ber.103 Although bio-DME has only half the energy content of conventional diesel, diesel engines can easily be retrofitted for bio-DME use. Well-to-wheel analyses showed that bio-DME was a little inferior to FT diesel for total fossil fuel substitu­tion and pollutant emissions.9192

After more than two decades of intensive molecular genetic research, S. cerevisiae remains the ethanologen of choice. Although industrial strains are genetically largely undefined, it is easily demonstrated that laboratory strains can function perfectly adequately for ethanol production, even for the complex process of manufacturing potable spirits with defined requirements for sensory parameters in the finished prod­uct such as volatiles, higher alcohols, and glycerol content.273 Nevertheless, it is more likely that applying the knowledge gained to the genomic improvement of hardy industrial Saccharomyces (or other Crabtree-positive yeast) strains with proven track records of fermenting very concentrated media to high volumetric yields of ethanol would generate highly suitable biocatalysts for the demanding tasks of growing and metabolizing sugars and oligosaccharides in lignocellulosic hydrolysates.

Even better would be the importing of a methodology developed in the world of industrial biotechnology, that is, “genome breeding,” as outlined by Kyowa Hakko Kogyo,[30] Tokyo, Japan. By comparing the whole genome sequence from the wild — type Corynebacterium glutamicum, the major producing organism for L-lysine and L-glutamate, with gene sequence from an evolved highly productive strain, it was possible to identify multiple changes; with these data, transforming a “clean” wild type to a hyperproducer of lysine was accomplished with only three specific and known mutations in the biosynthetic genes.274 This minimal mutation strain had dis­tinct productivity advantages over the industrial strains that had been developed by chance mutation over decades — a reflection of how many unwanted changes may (and did) occur over prolonged periods of random mutagenesis and selection in the twentieth century.275 Repeating such an exercise with any of the multitude of com­mercial alcohol-producing Saccharomyces yeasts would rapidly identify specific genomic traits for robustness and high productivity on which to construct pentose­utilizing and other capabilities for bioethanol processes.

A radically different option has been outlined in a patent application at the end of May 2007.276 Work undertaken at the J. Craig Venter Institute, Rockville, Maryland, defined a minimal set of 381 protein-encoding genes from Mycoplasma genitalium, including pathways for carbohydrate metabolism, nucleotide biosynthesis, phospho­lipid biosynthesis, and a cellular set of uptake mechanisms for nutrients, that would suffice to generate a free living organism in a nutritionally rich culture medium. Adding in genes for pathways of ethanol and/or hydrogen formation would result in a biofuels producer with maximum biochemical and biotechnological simplicity. The timeline required for practical application and demonstration of such a synthetic organism is presently unclear, although the research is funded by the DOE, under the genome projects of the Department’s Office of Science, with the target of develop­ing a novel recombinant cyanobacterial system for hydrogen production from water and a cellulosome system for the production of ethanol and/or butanol in suitable clostridial cells.277 The drawbacks to such an approach are that it would be highly dependent on the correct balance of supplied nutrients to the organism (with its lim­ited capabilities), requiring highly precise nutrient feeding mechanisms and a likely protracted optimization of the pathways to rival rates of product formation already attainable with older patented or freely available ethanologens.

A 14-million-gallon/year facility for 95% ethanol production from sugarcane molas­ses was technically the easiest production process to design and cost in the 1970s (table 5.3). To simplify even further, although coproducts (CO2 and fusel oils) were considered, no economic calculations were made for these; sale of the yeasts grown in

the fermentations were, however, included to effect a cost reduction of approximately 6.5%. The final calculated manufacturing price of 95% ethanol was $0.995/gallon, “a cost which is today very comparable to producing alcohol from ethylene.”12

Of the production costs, the raw material molasses was the dominating factor, accounting for 63% of the total — no exact geographical location for the hypotheti­cal facility was given but the quoted molasses price ($50/ton) was the 1978 summer average of molasses delivered to the East Coast and Midwest of the United States.