Direct comparisons of gas emissions resulting from the combustion of anhydrous ethanol, ethanol-gasoline blends, and gasoline are straightforward to perform but are poor indicators of the overall consequences of substituting ethanol for gasoline. Instead, from the early 1990s, full fuel-cycle analyses were performed to estimate gas emissions (projected beyond 2000) throughout the production process for bio­ethanol to gauge the direct and indirect consequences of gasoline replacement, including

• Changes in land use and the replacement of native species by energy crops

• Agricultural practices and the potential for utilizing agricultural wastes

• Materials manufacture and the construction of facilities

• Bioethanol production

• Transportation of feedstocks and bioethanol

• Fuel usage per mile driven

With the three major greenhouse gases, carbon dioxide (CO2), methane (CH4), and nitrous oxide (N2O), a major difference between corn-derived and cellulosic ethanol was immediately apparent.89 Figure 1.27 compares the projected CO2 emissions for a range of fuels; noncarbon-based fuels (i. e., electric vehicles powered using electricity generated by nuclear and solar options) were superior, whereas corn ethanol showed no net advantage. More recent estimates place corn ethanol production as giving modest reductions in greenhouse gas emissions, 12-14%.58 81 The GREET model of the Argonne National Laboratory indicates steeper reductions (figure 1.28).95

An unavoidable complication in any such calculation, however, is that they assume that harvested crops are from land already under cultivation; converting “native” ecosystems to biofuel production would inevitably alter the natural carbon balance and reduce the potential savings in greenhouse gas emissions.58

As with energy balance computations, including combined heat and power systems into cellulosic ethanol production displaces fossil fuel use and accounts for the reductions in greenhouse gas emissions.8188 In some corn ethanol production scenarios, the use of fossil fuels such as coal to power ethanol distillation plants and diesel to enable the long-distance transportation of corn can actually increase total greenhouse gas emissions.81 96 97 If the NEB is less than 1, the total ethanol production cycle would also increase net greenhouse emissions unless renewable energy sources rapidly supplanted fossil fuels in power generation.78,97,98 Corn sto­ver ethanol can, if its production process shares facilities with grain production, exhibit lower life cycle emissions than switchgrass-derived ethanol in projections up to 2010.99

For other priority pollutants, bioethanol production (even from cellulosic resources) and the use of bioethanol as a transportation fuel have ambiguous effects: increased nitric oxides, particulate matter, carbon monoxide, and volatile organic carbon but decreased sulfur oxides when measured in total in both urban and rural locations (figure 1.28). Combined fuel cycle and vehicle life cycle analyses also show major reductions in greenhouse gases but not in other emissions with cellulosic ethanol as an alternative fuel.100

For production sources other than sugarcane, therefore, the actual reductions in greenhouse gas emissions resulting from the adoption of biofuels may be considerably less than anticipated. One estimate for corn ethanol concluded that, by itself, fuel alcohol use in the United States would struggle to reduce transportation — dependent emissions by more than 10%.101 This study used the following data and arguments:

• For U. S. gasoline consumption of 460 x 109 L/year, corn ethanol replaced 0.8% of this while using 1% of the total cropland.

• To replace 10% of the gasoline consumption, corn ethanol would need to be produced on 12% of the total U. S. cropland.

• Corn ethanol only avoids 25% of the CO2 emissions of the substituted gaso­line emissions when the fossil fuel-dependent energy consumed to grow and process the corn is taken into account.

• Offsetting 10% of the CO2 emissions from gasoline consumption would require a fourfold higher production of corn ethanol, that is, from 48% of U. S. cropland.

• “A challenge for providing transportation fuels is to be able to substitute a joule of energy in harvestable biomass for a joule of primary energy in fossil fuel and to do this without significant fossil energy consumption. If a joule for joule consumption can be achieved, dedicated biomass plantations may be able to displace some 15% of the CO2 emissions expected from all uses of fossil fuels globally by 2030. For biofuels to replace more than about 15% of fossil-fuel CO2 emissions in 2030 will require a more rapid improvement in the energy efficiency of the global economy than is appar­ent from past trends in efficiency.”101

This pessimistic — or realistic — view of the quantitative impacts of biofuels on greenhouse gas scenarios in the twenty-first century was strongly reinforced by a mid-2007 study of alternatives to biofuels, specifically conserving and (if possible) extending natural forests, savannahs, and grasslands: compared with even the best biomass-to-biofuel case, forestation of an equivalent area of land was calculated to sequester at least twice the amount of CO2 during a 30-year period than the emis­sions avoided by biofuel use.102 As the authors concluded:

If the prime object of policy on biofuels is mitigation of carbon dioxide-driven global warming, policy makers may be better advised in the short term (30 years or so) to focus on increasing the efficiency of fossil fuel use, to conserve the existing forests and savannahs, and to restore natural forest and grassland habitats on cropland that is not needed for food.

Present national policies have, however, more diverse “prime object” aims and goals, including those of ensuring continuity of supply of affordable transportation fuels and (increasingly important on the geopolitical stage) establishing some degree of “energy independence.” Nevertheless, the assessment of potential savings in green­house gas emissions remains a powerful argument for the adoption of a considered, balanced raft of biofuels options.

4.1 THE IOGEN CORPORATION PROCESS AS A TEMPLATE AND PARADIGM

The demonstration process operated since 2004 is outlined in figure 4.1. In many of its features, the Iogen process is relatively conservative:

• Wheat straw as a substrate — a high-availability feedstock with a low lig­nin content in comparison with tree wood materials (figure 4.2)12

• A dilute acid and heat pretreatment of the biomass — the levels of acid are sufficiently low that recovery of the acid is not needed and corrosion problems are avoided

• Separate cellulose hydrolysis and fermentation with a single sugar substrate product stream (hexoses plus pentoses) for fermentation

• Cellulase breakdown of cellulose — Iogen is an enzyme producer

• A Saccharomyces yeast ethanologen — relatively ethanol-tolerant and engineered for xylose consumption as well as offering a low incidence of contamination, the ability to recycle the cells, and the option for selling on the spent cells for agricultural use1

In the first description of the process (written in and before July 1999), agricultural residues such as wheat straw, grasses, and energy crops (aspen, etc.) were equally “pos­sible” or a “possibility.”3 By the next appearance of the article in 2006[31] — and as dis­cussed in chapter 2, section 2.6 — cereal straws had become the substrates of choice. Lignin does not form a seriously refractory barrier to cellulase access with wheat straw; this renders organic solvent pretreatment unnecessary. More than 95% of the cellulosic glucose is released by the end of the enzyme digestion step, the remainder being included in the lignin cake that is spray-dried before combustion (figure 4.1).

The Iogen process is viewed as a sequential evolution of the bioethanol paradigm, no more complex than wet mill and dry mill options for corn ethanol production (figures 1.20 and 2.21), substituting acid pretreatment for corn grinding steps, and

adding on-site cellulase generation, the latter mostly as a strategy to avoid the costs of preservatives and stabilizer but possibly also to use a small proportion of the hydro­lyzed cellulose as a feedstock for the enzyme fermentation itself. Salient features of the technology were present in Canadian initiatives from the 1970s and 1980s. The Bio-hol process, financially supported by the Ontario Ministry of Energy and Energy, Mines, and Resources, Canada, opted for Zymomonas mobilis as the ethanologen and had established acid hydrolysis pretreatments for wheat straw, soy stalks, corn stover, canola stalks, pine wood, and poplar wood.4 For both Z. mobilis and S. cere — visiae, pretreated wheat straw had the distinct advantage of presenting far less of a toxic mixture to the producer organism (figure 4.3); methods for removing growth inhibitors from the biomass acid hydrolysates could reduce the effect by >20-fold.

Minimum Inhibitory Concentration (% w/v)

FIGURE 4.3 Growth of Z. mobilis on biomass hydrolysates. (Data from Lawford et al.4)

The Stake Company Ltd. was founded in 1973 to develop and market a process for biomass conversion to sugar streams for both biofuels and animal feeds as well as chemicals derived from lignin and hemicellulose.5 A continuous feedstock processing system was constructed to handle 4-10 tons of wood chips/hr and licensed to end — users in the United States and France.

Before 2004 (or 1999), moreover, more radical processes were examined in detail — including being upscaled to pilot plant operations — for lignocellulosic ethanol. These proposals included those to avoid the need for cellulase fermentations
independent of the main ethanolic fermentation as well as the use of thermophilic bacteria in processes that more closely resembled industrial chemistry than they did the traditional potable alcohol manufacture. Indeed, it is clear that Iogen consid­ered sourcing thermophilic bacteria[32] and nonconventional yeasts during the 1990s.3 The achieved reality of the Iogen process will, therefore, be used as a guide to how innovations have successfully translated into practical use — or have failed to do so — reviewing progress over (mostly) the last three decades and offering predictions for new solutions to well-known problems as the bioethanol industry expands geo­graphically as well as in production scale.

2.5.1 A Multiplicity of Hemicellulases

Mirroring the variety of polysaccharides containing pentoses, hexoses, or both (with or without sugar hydroxyl group modifications) collectively described as hemicelluloses (figure 1.23), hemicellulolytic organisms are known across many species and genera:15 [16]

Table 2.8 summarizes major classes of hemicellulases, their general sites of action, and the released products. Microorganisms capable of degrading hemicelluloses have, however, multiple genes encoding many individual hemicellulases; for exam­ple, Bacillus subtilis has in its completely sequenced genome at least 16 separate genes for enzymes involved in hemicellulose degradation.127

TABLE 2.8

Major Hemicellulases, Their Enzymic Sites of Action and Their Products

Hemicellulase EC number Site(s) of action Released products

Hemicelluloses comprise both linear and branched heteropolysaccharides. Endoxylanases fragment the xylan backbone, and xylosidases cleave the resulting xylan oligosaccharides into xylose; removal of the side chains is catalyzed by glucuron­idases, arabinofuranosidases, and acetylesterases — the action of these enzymes can limit the overall rate of hemicellulose saccharification because endoacting enzymes cannot bind to and cleave xylan polymers close to sites of chain attachment.128

Much of the fine detail of hemicellulase catalytic action is beginning to emerge and will be vital for directed molecular evolution of improved hemicellulase biocatalysts.129,130 Already, however, a thermostable arabinofuranosidase has been identified and shown to have a unique selectivity in being able to degrade both branched and debranched arabinans.131 Synergistic interactions among different microbial arabinofuranosidases have also been demonstrated to result in a more extensive degradation of wheat arabinoxylan than found with individual enzymes.132 The activity of biotech companies in patenting novel hemicellulase activities is evi­dent (table 2.9) in exploring hemicellulases from unconventional microbial sources, and deep-sea thermophilic bacteria from the Pacific have been shown to synthesize thermotolerant xylanases, to be active over a wide pH range, and to degrade cereal hemicelluloses.133

4.4.1 Continuous Fermentations for Ethanol Production

With enormous strides made in the development of high-alcohol fermentations pre­pared using high — and very-high-gravity media, and compatible with low operating temperatures, fast turnaround times, and high conversion efficiency from carbohy­drate feedstocks, what has the “traditional” ethanol industry achieved with fermen — tor hardware and process design and control?

Beginning in the late 1950s in New Zealand, the Dominion Breweries intro­duced and patented a novel “continuous” brewing process in which part of the fer­mented beer wort was recycled back to the wort of the start of the fermentation step (figure 4.8).149 Within two years, a rival continuous process had been announced (and patented) in Canada by Labatt Breweries, and independently conceived research was being published by the Brewing Industry Research Foundation in the United King — dom.150 By 1966, the concept had been simplified and reduced to a single-tower structure in which the bulk of the yeast cells were retained (for up to 400 hours) while wort might only reside in the highly anaerobic conditions in most of the tower for only 4 hours before a “beer” product emerged.[36] 151

However, despite much initial enthusiasm and the evolution of technologies into a family tree of “open” (where yeast cells emerge at rapid rate from the process), “closed” (where yeast cells are mostly retained), “homogenous” (approximating a stan­dard stirred-tank fermentor), and “heterogeneous” (with gradients of cells, substrates, and products across several vessels) systems devised for the continuous brewing of beer, very few were developed to the production scale and most became defunct. The innovations failed the test of four basic parameters of brewing practice:150

1. Commercial brewers offer multiple product lines and flexible production schedules to a marketplace that has become more sophisticated, discern­ing, and advertisement-influenced.

2. Continuous systems offer a fixed rate of beer production (and increasing flow rate tends to lead to wash out).

3. Only highly flocculent strains of yeast can be used.

4. Desired flavors and aromas cannot always be generated, whereas undesir­able levels of malodorous compounds are easily formed.

Moreover, there were contemporaneous developments in brewing technology that led, for example, to the marked reduction in malting and fermentation time in batch processes, the losses during malting and wort boiling, increased a-acid content and hops (and, consequently, reductions in hop usage), and the elimination of the prolonged “lagering” storage necessary for lager beer production (an increasing popular product line world­wide). Such incremental improvements were easy to retrofit in established (often vener­ably old) brewing facilities, which demanded mostly tried and trusted hardware for new sites. Other than in New Zealand, continuous brewing systems have proved generally unpopular — and the rise of globally recognized brands has resulted in the backlash of movements for traditional, “real ale,” locally produced (in microbreweries) options, continuing the trend of consumer choice against a perceived blandness in the output of the market leaders. Large multinationals, offering a branded product for global markets, remain the most likely to invest in “high-technology” brewing.

In contrast, industrial and fuel ethanol production is immune to such drivers, and by the late 1970s, the cold-shouldering of new technologies by potable alcohol producers had persuaded academic and research groupings of chemical engineers to focus on eth­anol as an alternative fuel with a renewed interest in highly engineered solutions to the demands for process intensification, process control, and cost reduction. For example, [37]

vessel, a high-productivity process was devised that incorporated sparging with O2 to support active cell growth over a prolonged period and bleed out the fermented broth to withdraw nonvolatile compounds of potential toxicity to the producing cells; for a 95% ethanol product, such technology offered a 50% reduction in production costs over batch fermentation.152

• A single-stage gas-lift tower fermentor with a highly flocculent yeast was designed to run with nearly total retention of the cell population and gener­ate a clear liquid effluent; analysis of the vapor-phase ethanol concentration in the headspace gas gave, via computer control, an accurate control of input and output flow rates.153

• A more straightforward use of laboratory-type continuous fermentors oper­ating on the fluid overflow principle and not requiring a flocculent yeast achieved a maximal ethanol yield (89% of the theoretical from carbohy­drate) at a low dilution rate (0.05/hr), showed 95% utilization of the inflow­ing sugars up to a dilution rate of 0.15/hr, and only suffered from washout at 0.41/hr; the fermentation was operated with a solubilized mixed substrate of sugars from Jerusalem artichoke, a plant species capable of a very high carbohydrate yield on poor soils with little fertilizer application.154

For large-scale fuel ethanol manufacture, however, it is another feature of the continuous process that has proved of widest application, that is, the use of multiple fermentors linked in series with (or without) the option of recycling the fermenting broth, sometimes described as “cascading” (figure 4.9).122 Several evolutionary variants of this process paradigm have been developed by the Raphael Katzen Associates and Katzen International, Cincinnati, Ohio, from design concepts for corn ethanol dating from the late 1970s.36 In the former USSR, batteries of 6-12 fermentors were linked in multistage systems for the production of ethanol from miscellaneous raw materials in 70 industrial centers, but, unlike the West, such advanced engineering was applied to other types of fermentation, including beer, champagne, and fruit wines, as well as fodder yeast (as a form of single-cell protein); these were supported by scientists at the All-Union Research Institute of Fermentation Products who developed sophisticated mathematical analyses, unfortunately in a literature almost entirely in Russian.155

Multistage fermentations have been merged with VHG media in laboratory sys­tems with 99% of consumption of media containing up to 32% w/v glucose.156 In such a continuously flowing system, ethanol yield from glucose increased from the first to the last fermentor in the sequence at all glucose concentrations tested (15-32% w/w); ethanol was, therefore, produced more efficiently in the later stages of the inhomoge­neous set of fermentations that are set up in quasi-equilibrium within the sequence of the linked fermentation vessels. This implies mathematical modeling to control pro­ductivity will be difficult in multistage processes because parameters such as ethanol yield from sugars will be variable and possibly difficult to predict with complex feed­stocks. In similar vein, a Chinese prototype with a working volume of 3.3 l achieved a 95% conversion of glucose to ethanol; although oscillations were observed in residual glucose, ethanol concentrations, and cell densities, some success was found in devis­ing models to predict yeast cell lysis and viability loss.157 In Brazil, attention was focused on the problems of running ethanol fermentations at high ambient (tropical)

temperatures, and a five-fermentor system was devised in which a temperature gra­dient of 8°C was established: high-biomass cultures were generated at up to 43°C, and a high-viability process with continuous ethanol production was demonstrable at temperatures normally considered supraoptimal for brewer’s yeast.158 A much simpler design, one transposed from brewery work in the United Kingdom, fermented sugar­cane juice at sugar concentrations up to 200 g/l in a tower with continuous recycling of a highly flocculent yeast strain; a constant dilution rate and pH (3.3) gave outflow ethanol concentrations of up to 90 g/l with conversion rates as high as 90% of the theoretical maximum.159 In France, the concept of separate compartments for growth and ethanologenesis was pursued in a two-stage fermentor with efficient recycling of the yeast cells; a steady state could be reached where the residual glucose concentra­tion in the second stage was close to zero.160

However described — continuous, cascaded, multistage, and others — sequen­tial fermentations offer a step change in fermentation flexibility that may be of great value for lignocellulosic feedstocks: different vessels could, for example, be oper­ated at higher or lower pH, temperature, and degree of aeration to ferment different sugars and could even accommodate different ethanologenic organisms. Figure 4.10 is a schematic of a process with split pentose and hexose sugar streams with five fermentors in the continuous cascaded sequence, the liquid stream that moves from fermentor to fermentor being in contact with a stripping gas to remove the ethanol

(thus avoiding the buildup of inhibitory concentrations); the process is a composite one based on patents granted to Bio-Process Innovation, West Lafayette, Indiana.161163 A low-energy solvent absorption and extractive distillation process recovers the etha­nol from the stripping gas, and the gas is then reused, but the same arrangement of fermentation vessels can be used with conventional removal of ethanol by distillation from the outflow of the final fermentor. The pentose and hexose fermentations could, moreover, employ the same recombinant organism or separate ethanologens — an example of this was devised by researchers in China who selected Pichia stipitis as the organism to ferment pentoses in an airlift loop tower while choosing S. cerevisiae for glucose utilization in an overflow tower fermentor for the residual glucose; with an appropriate flow rate, a utilization of 92.7% was claimed from the sugars prepared from sugarcane bagasse.95

Most of the freely available information regarding ethanologenic yeasts has been derived from “laboratory” strains constructed by research groups in academia; some of these strains (or variants thereof) have certainly been applied to industrial-scale fermentations for bioethanol production, but published data mostly refer to strains either grown in chemically defined media or under laboratory conditions (e. g., contin­uous culture) or with strains that can—even under the best available test conditions— accumulate very little ethanol in comparison with modern industrial strains used in potable alcohol or fuel alcohol production (figure 3.7). In addition, strains con­structed with plasmids may not have been tested in nonselective media, and plasmid survival in fermentations is generally speculative although genetic manipulations are routine for constructing “self-selective” plasmid-harboring strains where a chro­mosomal gene in the host is deleted or disrupted and the auxotrophic requirement is supplied as a gene contained on the plasmid.113

Nevertheless, benchmarking studies comparing “laboratory” and “industrial” strains constructed for pentose utilization have appeared, the industrial examples including genetically manipulated polyploid strains typical of S. cerevisiae “work­ing” strains from major brewers or wineries; accounts of engineering such strains for xylose utilization began to be published after 2002.114,115 A comparison of four labo­ratory and five industrial strains surveyed both genetically manipulated and geneti­cally undefined but selected xylose consumers (table 3.4).116 The industrial strains were inferior to the laboratory strains for the yields of both ethanol and xylitol from

xylose in minimal media. Resistance to toxic impurities present in acid hydrolysates of the softwood Norway spruce (Picea abies) was higher with genetically trans­formed industrial strains, but a classically improved industrial strain was no more hardy than the laboratory strains (table 3.4); similarly, while an industrial strain evolved by genetic manipulation and then random mutagenesis had the fastest rate of xylose use, a laboratory strain could accumulate the highest ethanol concentration on minimal medium with 50 g/l of each glucose and xylose. None of the strains had an ideal set of properties for ethanologenesis in xylose-containing media; long-term chemostat cultivation of one industrial strain in microaerobic conditions on xylose as the sole carbon source definitely improved xylose uptake, but neither ethanol nor xylitol yield. Three of the industrial strains could grow in the presence of 10% solu­tions of undetoxified lignocellulose hydrolysate; the most resistant strain grew best (at 4.3 g/l as compared with 3.7-3.8 g/l) but had a marginally low ethanol production (16.8 g/l as compared with 16.9 g/l), perhaps because more of the carbon substrate was used for growth in the absence of any chemical limitation.

The industrial-background strain TMB 3400 (table 3.4) had no obvious meta­bolic advantage in anaerobic batch fermentations with xylose-based media when compared with two laboratory strains, one catabolizing xylose by the XR/XDH/XK pathway, the other by the fungal XI/XK pathway (figure 3.8).101 In the presence of undetoxified lignocellulose hydrolysate, however, only the industrial strain could grow adequately and exhibit good ethanol formation (figure 3.8).

Industrial and laboratory strains engineered for xylose consumption fail to metabolize L-arabinose beyond L-arabitinol.116 With laboratory and industrial strains endowed with recombinant xylose (fungal) and arabinose (bacterial) pathways tested in media containing glucose, xylose, and arabinose, the industrial strain accumulated higher concentrations of ethanol, had a higher conversion efficiency of ethanol per

unit of total pentose utilized, and also converted less xylose to xylitol and less arabi — nose to arabinitol — although 68% of the L-arabinose consumed was still converted only as far as the polyol.117

Interactions between hexose and pentose sugars in the fermentations of lignocel- lulose-derived substrates has often been considered a serious drawback for ethanol production; this is usually phrased as a type of “carbon catabolite repression” by the more readily utilizable hexose carbon source(s), and complex phenotypes can be gen­erated for examination in continuous cultures.118 In batch cultivation, xylose supports slower growth and much delayed entry into ethanol formation in comparison with glucose.119 An ideal ethanologen would co-utilize multiple carbon sources, funneling them all into the central pathways of carbohydrate metabolism — ultimately to

pyruvic acid and thence to acetic acid, acetaldehyde, and ethanol (figures 3.2 and 3.4). No publicly disclosed strain meets these requirements. An emerging major challenge is to achieve the rapid transition from proof-of-concept experiments in synthetic media, using single substrates and in the absence of toxic inhibitors, to demonstra­tions that constructed strains can efficiently convert complex industrial substrates to ethanol.120 In addition to the key criteria of high productivity and tolerance of toxic impurities, process water economy has been emphasized.121

Integration of genes for pentose metabolism is becoming increasingly routine for S. cerevisiae strains intended for industrial use; different constructs have quantitatively variable performance indicators (ethanol production rate, xylose consumption rate, etc.), and this suggests that multiple copies of the heterologous genes must be further optimized as gene dosages may differ for the individual genes packaged into the host strain.122 Such strains can be further improved by a less rigorously defined methodology using “evolutionary engineering,” that is, selection of strains with incremental advan­tages for xylose consumption and ethanol productivity, some of which advantages can be ascribed to increases in measurable enzyme activities for the xylose pathway or the pentose phosphate pathway and with interesting (but not fully interpretable) changes in the pool sizes of the intracellular pathway intermediates.123 124 Efficient utilization of xylose appears to require complex global changes in gene expression, and a reexamina­tion of “natural” S. cerevisiae has revealed that classical selection and strain improve­ment programs can develop yeast cell lines with much shorter doubling times on xylose as the sole carbon source as well as increased XR and XDH activities in a completely nonrecombinant approach.125 This could easily be applied to rationally improve yeast strains with desirable properties that can be isolated in the heavily selective but artifi­cial environment of an industrial fermentation plant — a practice deliberately pursued
for centuries in breweries and wineries but equally applicable to facilities for the fer­mentation of spent sulfite liquor from the pulp and paper industry.126

Defining the capabilities of both industrial and laboratory strains to adapt to the stresses posed by toxic inhibitors in lignocellulose hydrolysates remains a focus of intense activity.127130 Expression of a laccase (from the white rot fungus, Trametes versicolor) offers some promise as a novel means of polymerizing (and precipitat­ing) reactive phenolic aldehydes derived from the hydrolytic breakdown of lignins; S. cerevisiae expressing the laccase could utilize sugars and accumulate ethanol in a medium containing a spruce wood acid hydrolysate at greatly increased rates in comparison with the parental strain.131 S. cerevisiae also contains the gene for phenylacrylic acid decarboxylase, an enzyme catalyzing the degradation of ferulic acid and other phenolic acids; a transformant overexpressing the gene for the decar­boxylase utilized glucose grew up to 25% faster, utilized mannose up to 45% faster, and accumulated ethanol up to 29% more rapidly than did a control transformant not overexpressing the gene.132

BIOFUEL: PRESENT STATUS AND FUTURE PROSPECTS

The developments of sugar-derived ethanol as a major transportation fuel in Brazil and corn-derived ethanol as a niche (but rapidly growing) market in the United States were conditioned by different mixes of economic and environmental imperatives from the early 1970s to the present day. Together and separately, they have been criticized as unsuitable for sustainable alternatives to gasoline in the absence of tax incentives; highly integrated production processes, with maximum use of coproducts and high degrees of energy efficiency, are mandated to achieve full economic competitiveness. Although technically proven as a technology, the scope of ethanol production from food crops (primarily sugar and corn) is limited by agricultural and geographical factors, and only cellulosic sources offer the quantita­tive availability to significantly substitute for gasoline on a national or global basis. Even for that goal, however, bioethanol is only one player in a diverse repertoire of strategic and tactical ploys and mechanisms — from carbon capture and storage to hydrogen-utilizing fuel cells — with which to change the oil economy and reduce dependence on fossil fuels.103

Both corn — and sugarcane-derived ethanol have — unarguably but not irre­versibly — emerged as industrial realities.104 Even detailed questions about their NEBs and the implications of their use in mitigating greenhouse gas emissions have become unavoidable on Web site discussion forums. A major article in the October issue of National Geographic magazine accepted the marginal energy gain in corn ethanol production and the disappointingly small reduction of total CO2 emissions in the total production/use cycle but noted the much clearer benefits of sugarcane ethanol.105 Two quotes encapsulate the ambiguous reactions that are increasingly evident as the public debate over biofuels spreads away from scientific interest groups to embrace environmentalist and other “lobbies”:

It’s easy to lose faith in biofuels if corn ethanol is all you know, and

If alcohol is a “clean” fuel, the process of making it is very dirty… especially the

burning of cane and the exploitation of cane workers.

All forms of ethanol produced from plant substrates are, however, best viewed as “first-generation” biofuels, operating within parameters determined by (in varying allocated orders): the internal combustion engine, mass personal transport, and the global oil industry.105 Together with biodiesel (i. e., chemically esterified forms of the fatty acids present in vegetable oils — see chapter 6, section 6.1), they have proven potential for extending the availability of gasoline blends and are affordable within the budgets accepted by consumers in the late twentieth and early twenty — first centuries while helping to reduce greenhouse gas emissions and (with some technologies, at least) other atmospheric pollutants. It is, however, likely that different countries and supranational economic groupings (e. g., the European Union) will value differently energy security, economic price, and ecological factors in the face of fluctuating oil prices and uncertain mid — to long-term availabilities of fossil fuels.106 Fermentation products other than ethanol (e. g., glycerol, butanol) and their thermochemical conversion to synthesis gas mixtures to power electricity generation are second-generation technologies, whereas biohydrogen, fuel cells, and microbial fuel cells represent more radical options with longer lead times.107108

Sugar — and corn-derived ethanol production processes were simply extrapolated from preexisting technologies, and to varying extents, they reflect the limitations of “add-on” manufacturing strategies.109110 As one example of the National Geographic writer’s anticipated “breakthrough or two,” lignocellulosic ethanol only emerged as a commercial biofuel reality in 2004, and biotechnology is crucial to its establishment as a major industry.105111 For that reason, and because of the uniquely global potential of this nonfood sector of the bioethanol supply chain, the microbial biotechnology of cellulosic ethanol will now be considered at length, to provide not only a snapshot of commercially relevant contemporary science but also to extrapolate existing trends in the development of the scientific base.

In the calculations inherent in the data for figure 4.2, some interesting conclusions are reached. Although wheat straw has undoubted advantages, other feedstocks out­perform wheat straw for some key parameters:

• Wheat straw has a lower gravimetric ratio of total carbohydrate (cellulose, starch, xylan, arabinan) to lignin than barley straw, corn stover, switch — grass, or even wheat chaff.

• Wheat straw has a lower cellulose:lignin ratio than all of these nonsoftwood sources (with the exception of switchgrass).

• Of the seven quoted examples of lignocellulosic feedstocks, wheat chaff and switchgrass have the highest total pentose (xylan and arabinan) con­tents — quoted as an important quantitative predictor for ethanol yield from cellulose because less cellulase is required (or, conversely, more of the cellulose glucose is made available for fermentation).1,2

An important consideration included in Iogen’s deliberations on feedstock suit­ability was the reproducible and predictable supply of wheat straw. The USDA Agricultural Service also itemized sustainable supply as one of their two key fac­tors for biomass feedstocks, the other being cost-effectiveness.6 Financial models indicate that feedstocks costs are crucial, and any managed reduction of the costs of biomass crop production, harvesting, and the sequential logistics of collec­tion, transportation, and storage before substrate pretreatment will all impact the viability of biofuel facilities — economic aspects of feedstock supply chains are discussed in the next chapter. The Energy Information Administration has con­structed a National Energy Modeling System to forecast U. S. energy production, use, and price trends in 25-year predictive segments; the biomass supply schedule includes agricultural residues, dedicated energy crops, forestry sources, and wood waste and mill residues, and wheat straw (together with corn stover, barley straw, rice straw, and sugarcane bagasse) is the specified component of the agricultural residue supply.7

A brief survey will, therefore, be made of wheat straw and other leading candi­date lignocellulosics, with special emphasis on how different national priorities place emphasis on different biomass sources and on what evolving agricultural practices and processing technologies may diversify bioethanol facilities on scales equal to and larger than the Iogen demonstration facility.

The importance of including hemicellulosic sugars in the conversion of lignocellulosic feedstocks to ethanol to ensure process efficiency and an economic base for biofuel production has often been emphasized.134135 Given that hemicellulosic sugars constitute a fermentable resource equal to approximately 50% of the glucose residues present in the cellulose in most plant species (table 1.5), this is an unsurprising conclusion.

Thermochemical and acid-catalyzed pretreatments of lignocellulosic biomass materials extensively degrade hemicelluloses (see above, section 2.3.2); depending

on the pretreatment method and the feedstock. However, hemicellulose solubilization may be as high as 100% or as low as 10%, and the hemicellulose sugars may be pres­ent primarily as monomers (xylose, arabinose, etc.) or as oligomers.60 A detailed investigation of destarched corn fiber as a starting material, initially extracted with hot water, showed that 75% of the total hemicellulose could be easily extracted; if enzyme preparations generated by growing Hypocrea jecorina and Aspergillus niger (containing xylanase, P-xylosidase, and feruloyl esterase as well as cellulase) were then used, the recovery of total xylan as xylose increased greatly (from <15% to >50%), and the arabinose yield also improved, although from a much higher starting point of more than 50%; total recoveries of hemicellulosic sugars such as arabinose and xylose reached 80% when supplementations with commercial glucoamylase, P-glucosidase, and feruloyl esterase were included.136 Similarly, a xylose yield of 88% of the theoretical — as the free monosaccharide xylose — was achieved from pretreated corn stover by supplementing cellulase with xylanase, pectinase, and P-glucosidase activities.122 Commercial cellulase preparations contain variable but often high activities of hemicellulases; this may have adventitiously contributed to the production of hemicellulose sugars in lignocellulosic materials processed for ethanol production.137,138

Beyond ethanol production, the modern mainstream fermentation industry manufac­turing primary metabolites including vitamins and amino acids, secondary metab­olites (including antibiotics), and recombinant proteins almost invariably opts for fed-batch fermentation technologies and has invested much time and expertise in devising feeding strategies for carbon sources (including sugars, oligosaccharides, and amylase-digested starches) to operate at a minimum concentration of free sug­ars and avoid carbon catabolite repressions. Some large-scale processes are run to vanishingly small concentrations of free glucose, and the feed rate is regulated not by direct measurement but indirectly by effects of transient glucose accumulation on physical parameters such as pH (responding to acid accumulation during glucose overfeeding) or the trends in dissolved O2 concentration.164

Yeast (S. cerevisiae in its “baker’s yeast” guise) cells are one of the three principal production platforms for recombinant proteins for the biopharmaceuticals market, the others being E. coli and mammalian cell cultures: of the 10 biopharmaceutical protein products achieving regulatory approval in the United States or the European Union during 2004, seven were produced in mammalian cell lines, two in E. coli, and the tenth in S. cerevisiae.165 In such cell systems, ethanol formation is either to be avoided (as a waste of glucose) or carefully regulated, possibly as a means of feedback control to a complex and variable sugar feed to high cell densities where O2 supply is critical to maintain anabolic, biosynthetic reactions rather than simple

fermentation.166,167

For fuel ethanol, in contrast, very high rates of sugar consumption and ethanol production are mandatory for competitive, commercial production processes. Tem­perature is, as always, an important parameter: under European conditions, 30°C is optimal for growth and 33°C for ethanol production.168 Rather than aiming at micro­aerobic conditions, a high-aeration strategy is beneficial for stabilizing a highly viable cell mass capable of high ethanol productivity.168 Glycerol accumulates as a major coproduct, but this waste of sugar metabolism can be minimized by several options for fermentation management: [38]

accumulation may (in addition to a role in osmotolerance) offer some degree of temperature protection to ethanol-forming yeast cells168

• High-aeration regimes greatly reduce glycerol accumulation169

• Maintaining a high respiratory quotient (the ratio of CO2 produced to O2 consumed) results in a high ethanol-to-glycerol discrimination ratio when the online data are used to feedback control the inlet sugar feed rate170

In addition to cane sugar, fed-batch technology has been used to produce ethanol from sugarcane molasses with an exponentially decreasing feed rate.171 The detailed mathematical model advanced by the Brazilian authors, incorporating the feed rate profile and two further process variables, is probably too complex for small-scale fermentation sites but well within the capabilities of facilities operating (and manag­ing) large, modern fermentors.

The bacterial ethanologen Z. mobilis is most productive both for biomass forma­tion and ethanol production from glucose when feeding avoids the accumulation of high concentrations of glucose; an important finding from this work was that attempts to regulate a constant glucose concentration do not optimize the process because of the complex relationship between specific growth rate and glucose supply.172 In gen­eral, experience in the fermentation routes to producing fine chemicals highlights the importance of accurately monitoring analyte levels inside fermentors to avoid exces­sive accumulations (or depletions) of key substrates and nutrients and triggering repres­sion mechanism and the appearance of metabolic imbalances, all of which negatively impact on process economics.173 In early 2007, a major collaboration was announced to provide automated, near real-time online monitoring of commercial fuel ethanol fermentations using proprietary methods to sample high-solids and highly viscous fer­mentation broths.174 A spectrum of measurements was included in the design remit, including methodologies for ethanol, sugars, and organic acids. Because fed-batch processes are widely considered to be the favored route to the contemporary yeast cell limit of 23% v/v ethanol production, bioprocess management using more sophisticated tools to ensure a steady and slow release of glucose and other monomeric sugars are a major future milestone for industrial-scale bioethanol production.121

Serious development of yeasts other than S. cerevisiae has been muted; this is partly because of the ease of genetic transformation of S. cerevisiae and closely related strains with bacterial yeast shuttle vectors. Among nonconventional yeasts, C. she — hatae has, however, properties highly desirable for bioethanol production from lig — nocellulosic substrates:133

• Ethanol production is more efficient from a mixture of glucose and xylose than from either sugar alone.

• Ethanol production can be demonstrated at elevated temperatures (up to 45°C).

• Ethanol formation from xylose is not affected by wide variation in the xylose concentration in the medium.

• Ethanol can be produced from rice straw hemicellulose hydrolysates.

With a straightforward liquid hot water pretreatment of alfalfa fibers, C. shehatae could produce ethanol at a concentration of 9.6 g/l in a batch fermentation with a conversion efficiency of 0.47 g/g sugar consumed; hemicellulose utilization was, however, poor because of the presence of inhibitors.134 The methylotrophic yeast Hansenula poly — morpha can, on the other hand, ferment xylose as well as glucose and cellobiose; this species is thermotolerant, actively fermenting sugars at up to 45°C and with a higher ethanol tolerance than P. stipitis (although less than S. cerevisiae); a vitamin B2 (ribo — flavin)-deficient mutant exhibited increased ethanol productivity from both glucose and xylose under suboptimal riboflavin supply and the consequent growth restriction.135

P. stipitis, the host organism for genes of a xylose metabolism pathway success­fully expressed in S. cerevisiae, has been developed as an ethanologen by a research group at the University of Wisconsin, Madison, since the early 1990s.136 Part of this work was the development of a genetic system for P. stipitis, which was used to endow the yeast with the ability to grow and produce ethanol anaerobically.

P. stipitis is Crabtree-negative and is poorly productive for ethanol. S. cerevisiae derives its ability to function anaerobically from the presence of a unique enzyme, dihydroorotate dehydrogenase (DHOdehase), converting dihydroorotic acid to orotic acid in the pyrimidine biosynthetic pathway for nucleic acids; in S. cerevisiae, DHO — dehase is a cytosolic enzyme catalyzing the reduction of fumaric acid to succinic acid,[25] and the enzyme may constitute half of a bifunctional protein with a fuma — rate reductase or be physically associated with the latter enzyme inside the cell.137 Expression of the S. cerevisiae gene for DHOdehase (ScURAl) in P. stipitis enabled rapid anaerobic growth in a chemically defined medium with glucose as sole carbon source when essential lipids were supplied; 32 g/l of ethanol was produced from 78 g/l glucose in a batch fermentation.138

In mixtures of hexoses and pentoses, xylose metabolism by P. stipitis is repressed, whereas glucose, mannose, and galactose are all used preferentially, and this may limit the potential of the yeast for the fermentation of lignocellulosic hydrolysates; neither cellobiose nor L-arabinose inhibits induction of the xylose catabolic path­way by D-xylose.139 Ethanol production from xylose is also inhibited by the CaSO4 formed by the neutralization of sulfuric acid hydrolysates of lignocellulosic mate­rials with Ca(OH)2, whereas Na2SO4 (from NaOH) had no effect on either xylose consumption or ethanol production and (NH4)2SO4 (from NH4OH) reduced growth but enhanced the xylose utilization rate, the rate of ethanol production, and the final ethanol concentration.140 P. stipitis has been shown to produce ethanol on an array of lignocellulosic substrates: sugarcane bagasse, red oak acid hydrolysate, wheat straw, hardwood hemicellulose hydrolysate, and corn cob fractions.141145

Strains of the thermotolerant Kluyveromyces yeasts are well known to modern biotechnology as vehicles for enzyme and heterologous protein secretion.146 K. marx — ianus is one of the extraordinary biodiversity of microbial flora known to be present in fermentations for the spirit cachaga in Brazil (chapter 1, section 1.2); more than 700 different yeast species were identified in one distillery during a season, although S. cerevisiae was the usual major ethanologen except in a small number of cases where Rhodotorula glutinis and Candida maltosa predominated.147 Strains of K. marxia — nus isolated from sugar mills could ferment glucose and cane sugar at temperatures up to 47°C and to ethanol concentrations of 60 g/l, although long fermentation times (24-30 hr) and low cell viability were operational drawbacks.148 In another study, all eight strains that were screened for D-xylose use were found to be active and one K. marxianus strain was capable of forming ethanol at 55% of the theoretical maximum yield from xylose.149 A medium based on sugarcane molasses was fermented to a final ethanol level of 74 g/l at 45°C, but osmotic stress was evident at high concentra­tions of either molasses or mixtures of sucrose and molasses.150 Brazilian work has shown that K. marxianus is strictly Crabtree-negative, requiring (at least in labora­tory chemostat experiments) the O2 supply to be shut down for ethanol to be formed; a high tendency to divert sugars via the oxidative pentose phosphate pathway may be the major obstacle to this yeast as an ethanologen, but metabolic engineering could be applied to redirect carbon flow for fermentative efficiency.151

Some other yeast species are considered in the next chapter when microbes natu­rally capable of hydrolyzing polysaccharides (or engineered to do so) as well as fer­menting the resulting sugars are considered.