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

The case of the mostly widely applauded biofuel scheme to date, that of sugar­cane ethanol in Brazil, also has major doubts from the environmental perspective, although the first decade (1976-1985) of the program probably achieved a reason­able soil balance by recycling fermentor stillage as fertilizer, a valuable source of minerals, particularly potassium.20 36 With the great expansion of the industry sub­sequently, however, significant pollution problems have emerged. The volume of stillage that can be applied varies from location to location, and in regions with near-surface groundwater, much less stillage can be applied without contaminat­ing the water supply.36 In the case of the Ipojuca river in northeast Brazil, sugar cultivation and adjacent ethanol production plants use stillage extensively for both fertilization and irrigation, and this has led to water heating, acidification, increased turbidity, O2 imbalance, and increased coliform bacteria levels.98 The authors of this joint German-Brazilian study urged that a critical evaluation be made of the pres­ent environmental status of the sugar alcohol industry, focusing on developing more environmentally friendly cultivation methods, waste-reducing technologies, and water recycling to protect the region’s water resources.

The preservation of surface and groundwater in Brazil in general as a con­sequence of the sugar alcohol industry’s activities and development was ranked “uncertain, but probably possible” (table 5.17).99 Sugarcane plantations have been found to rank well for soil erosion and runoff criteria in some locations in Sao Paolo state, although the experimental results date from the 1950s (table 5.18). A much more recent study included in the second, Dutch-Brazilian report showed much poorer results for sugarcane in comparison with other monoculture crops (fig­ure 5.9). Nevertheless, although Brazilian sugarcane alcohol (viewed as an indus­trial process) makes massive demands on the water supply (21 m3/tonne of cane input), much of this water can (in principle) be recycled; in addition, Brazil enjoys such a large natural supply of freshwater from its eight major water basins (covering an area of 8.5 million km2) that the ratio of water extracted to supply is, on a global basis, exceedingly small: approximately 1%/annum, equivalent to 30-fold less than comparable data for Europe. Local seasonal shortages may, however, occur, and two of the four main sugar production regions have relatively low rainfalls (figure 5.10). Although sugar cultivation has mainly been rain-fed, irrigation is becoming more common.

Annual Soil Losses by Erosion and Runoff in Experimental Stations in Brazil

North and
northeast (sugar)

1.0 1.5

Rainfall (mm/km2/year)

FIGURE 5.10 Annual rainfall in main sugar-producing and other regions of Brazil. (Data from Smeets et al.99)

the enormous consumption levels of the Global North will not lead the Brazilian countryside out of poverty or help attain food sovereignty for its citizens.”100 On the other hand, to achieve poverty alleviation and the eradication of social exclusion and with support from environmentalists, Brazil proposed the Brazilian Energy Initiative at the 2002 World Summit on Sustainable Development (Johannesburg, South Africa) aiming at the establishment of global targets and timeframes of min­imum shares of energy from renewable sources.101 Headline figures for the global numbers of malnourished people known to international agencies are another datum point with a large uncertainty: from below 1 billion to 3.7 billion.3895 As an economist from the Earth Policy Institute was quoted as saying: “The competition for grain between the world’s 800 million motorists to maintain their mobility and its two billion poorest people who are simply trying to stay alive is emerging as an epic issue.”102

Brazil became the global leader in ethanol exports in 2006, exporting 19% (3 bil­lion liters) of its production — 1.7 billion liters of which were imported by the United States — and plans to export 200 billion liters annually by 2025, increasing sugar­cane planting to cover 30 million hectares.100 Sugar for ethanol will increasingly be viewed by nations without a strong industrial base but with suitable climatic condi­tions for sugarcane growth as a cash crop, in exactly the manner that Brazil regards coffee or soybeans; the example provided by Brazil in creating rural employment at low cost, reducing the economic burden of oil imports, and developing national industrial infrastructure will be one difficult to resist, especially if major sugar pro­ducers, including Brazil, India, Cuba, Thailand, South Africa, and Australia, unite to create an expanding alternative fuel market with sugar-derived ethanol.36 South Africa, for example, has a great and acknowledged need to improve its sugarcane

economy, where 97% of its sugarcane growers are small scale, achieving only a quarter of the productivity realized by commercial operators; sugar is produced in a surplus, most of which is exported, but a national plan to encourage biofuels usage is in place, and a first ethanol plant is planned for construction by a South African sugar producer[54] in neighboring Mozambique.103

Academic economists and agronomists are calling (and will continue to call) for an informed debate about land use in the context of increasingly large areas of highly fertile or marginal land being reallocated for energy crops.104 Although there is good evidence that sugarcane-derived ethanol in Brazil shows the highest agri­cultural land efficiency in both replacing fossil energy for transportation and avoid­ing greenhouse gas emissions (figure 5.11), impacts on acidification and human and ecological toxicity and deleterious environmental effects occurring mostly during the growing and processing of biomass are more often ranked as unfavorable than favorable in surveys.105

The principal economic drivers toward greater biofuel production in develop­ing economies are, however (and paradoxically), those widely accepted programs to reduce greenhouse gas emissions, increase energy security, and move to a scientifi­cally biobased economy by promoting the use of biofuels (table 5.19). If a new orga­nization of ethanol exporting countries, mostly in the Southern Hemisphere, arises to make up any shortfall in the production of endogenous biofuels in major OECD economies, only a sustained effort to require and enforce agronomically sound and environmentally safe practices on the part of those net importers will provide

200

5.12 The impact of fuel economy on projected demand for ethanol in various gasoline blends. (Data from Morrow et al.76)

Selected Policies on Light-Duty Vehicle Fuel Economy

• Shifting our reliance on petroleum products to biobased products that gen­erally have fewer harmful environmental effects

When another principle is added — strengthening rural economies and increasing demand for agricultural commodities — the main issues of the political agenda that has emerged post-2000 in both the United States and OECD economies in general are clear. There is one final argument, however, and one that commenced in the 1950s, that, instead of rendering the question of economic price of biofuels irrel­evant, reformulates the question to ask: how will biofuels affect the cost of living and personal disposable income in the twenty-first century?

Vast caverns of CO2-absorbing bacterial fermentations producing high carbon-con­tent products with immediate human use — including bacterial cellulose as fiber, single-cell protein, or bioplastics — may be industrial realities for the later years of the present century but perhaps a more compressed timescale should occupy a high priority on the biofuels and climate change agenda. Over the coming 25 years, “hard

truths” about the global energy future will be (or are) unavoidable, and the role of biomass and other renewables in the emerging technological mix is a key issue.100

Any individual’s ranking of the immediate challenges that could be met by biotechnology is biased and partial but a useful departure point may be the prior­ity list of discussion items in a major international conference on biofuels held in 2007.

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

Intended for Biofuel Production

With a lignocellulosic platform for bioethanol production, one obvious target is (as just discussed) the management of energy crop productivity to maximize the capture of solar energy and atmospheric CO2; the chemical composition of the bio­mass is, however, of great practical significance for the industrial bioprocessing of

feedstocks:302,303

1. Developing crop varieties with reduced lignin contents (especially with softwoods)

2. Crops with increased cellulose and, arguably, hemicellulose contents

3. Plants with the increased capability to degrade cellulose, hemicellulose, and lignin — after harvest (i. e., in a controlled manner capable of minimiz­ing biomass pretreatment)

Of these, modifying lignin content has been the most successful — classical genetics suggests that defining quantitative traits and their genetic loci is relatively easy, and (even better) some of these loci are those for increased cellulose biosynthesis.304 As collateral, there is the confidence-building conclusion that lignin contents of commer­cial forest trees have been reduced to improve pulping for the paper industry; the genetic fine-tuning of lignin content, composition, or both is now technically feasible.305

Reductions in plant lignin content have been claimed using both single — and multiple-gene modifications (figure 4.16):

• Down-regulating either of the initial two enzymes of lignin biosynthesis, phenylalanine ammonia lyase and cinnamate 4-hydroxylase (C4H), reduces lignin content and impairs vascular integrity in the structural tissues of

plants.305

• Deletion of the second activity of the bifunctional C4H enzyme, coumarate 3-hydroxylase, results in reduced lignin deposition.306

• Later enzymes in the lignin pathway were considered to be less amena­ble for inhibiting lignification but multiple-gene down-regulation could

be effective.307,308

• Inactivating O-methyltransferase activity with an aspen gene incorporated into a transmissible plasmid in the antisense orientation reduced lignin for­mation in Leucaena leucocephata[45] by 28%, increased monomeric phenolic levels, and increased the cellulose content by 9% but did not visibly affect the plant phenotype.309

Are “lignin-light” plants biologically viable for commercial cultivation? Altered stem lignin biosynthesis in aspen has a large effect on plant growth, reducing total leaf area and resulting in 30% less total carbon per plant; root growth was also

4-OH’Coumaric ||
acid

СОСоА

I

СН

4’OH’COumarylCoA II

4’OH’Coumarylaldehyde 4-OHcoumaryl alcohol

FIGURE 4.16 Outline of biosynthesis of lignin precursors: PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; C3H, 4-coumarate 3-hydroxylase; COMT, caffeate O-methyltransferase; CCoAOMT, caffeoyl-CoA O-methyltransferase; CCR, cinnamoyl-CoA reductase; CAD, cinnamyl alcohol dehydrogenase; F5H, ferulate 5-hydroxylase. (After Hertzberg et al.312)

compromised.310 Vascular impairment can lead to stunted growth.307 On the other hand, aspen wood in reduced-lignin transgenics was mechanically strong because less lignin was compensated for by increased xylem vessel cellulose.308 Smaller plants may be grown, as energy crops, in denser plantations; alternatively, plants with reduced stature may be easier to harvest, and various practical compromises between morphology and use can be imagined — this can be seen as analogous to the introduction of dwarfing rootstocks for fruit trees that greatly reduced plant height and canopy spread and facilitated manual and mechanical harvesting.

Also without obvious effects on plant growth and development was the introduc­tion and heterologous expression in rice of the gene from Acidothermus cellulolyticus encoding a thermostable endo-1,4-P-glucanase; this protein constituted approximately 5% of the total soluble protein in the plant and was used to hydrolyze cellulose in ammonia fiber explosion-pretreated rice and maize.311 More ambitiously, enzymes of polysaccharide depolymerization are being actively targeted by plant biotech compa­nies for new generations of crops intended for biofuels. A large number of genes are under strict developmental stage-specific transcriptional regulation for wood forma­tion in species such as hybrid aspen; at least 200 genes are of unknown function, pos­sibly undefined enzymes and transcription factors, but this implies that heterologous glucanases and other enzymes could be produced during plant senescence to provide lignocellulose processing in plants either before the preparation of substrates for con­ventional ethanol fermentation or in solid-phase bioprocesses (section 4.6.2).312

Glycerol was produced on an industrial scale by fermentation in the first quarter of the twentieth century (especially during World War I) but then declined, unable to compete with chemical synthesis from petrochemical feedstocks.106 A similar historical fate occurred with the ABE fermentation-producing “solvents,” that is, acetone, butanol, and ethanol in various proportions. Beginning (as with fuel etha­nol) with the oil crises of the 1970s, renewed interest was evinced in the technology, aided greatly by the accelerating advance of microbial physiology and genetics at that time.115116 The microbial species capable of this multiproduct biosynthesis are clostridia, which also have remarkable appetites for cellulosic and hemicellulosic polymers, able to metabolize hexose sugars and pentoses (usually, both xylose and arabinose).117,118 This again parallels the drive to produce ethanol from lignocellu — losic biomass substrates (chapter 3, section 3.3.2.5). It came as no surprise, there­fore, when the neologism “biobutanol” (for и-butanol, C4H9OH) appeared. DuPont, Wilmington, Delaware, and British Petroleum are the companies most associated with the development of butanol as an advanced biofuel and which aim to market biobutanol by the end of 2007; according to the DuPont publicity material (www2. dupont. com), biobutanol’s advantages are persuasive:

• Butanol has a higher energy content than ethanol and can be blended with gasoline at higher concentrations for use in standard vehicle engines (11.5% in the United States, with the potential to increase to 16%).

• Suitable for transport in pipelines, butanol has the potential to be intro­duced into gasoline easily and without additional supply infrastructure.

• Butanol/gasoline mixtures are less susceptible to separate in the presence of water than ethanol/gasoline blends, demanding no essential modifications to blending facilities, storage tanks, or retail station pumps.

• Butanol’s low vapor pressure (lower than gasoline) means that vapor pres­sure specifications do not need to be compromised.

• Production routes from conventional agricultural feedstocks (corn, wheat, sugarcane, beet sugar, cassava, and sorghum) are all possible, supporting global implementation.

• Lignocellulosics from fast-growing energy crops (e. g., grasses) or agricul­tural “wastes” (e. g., corn stover) are also feasible feedstocks.

The principal hurdles to process optimization were in manipulating cultures and strains to improve product specificity (figure 6.11) and yield and in reduc­ing the toxicity of butanol and O2 (the fermentation must be strictly anaerobic)

Carbon Source

FIGURE 6.11 Variation in butanol production with two strains of Clostridium acetobutylicum grown on six different carbon sources. (Data from Singh and Mishra.118)

to producing cells.118119 Notable among advances made in the last decade are the following:

• Isolation of hyperproducing strains — Clostridium beijerinckii BA101 expresses high activities of amylase when grown in starch-containing media, accumulating solvents up to 29 g/l and as high as 165 g/l when adapted to a fed-batch fermentation with product recovery by pervaporation using a silicone membrane.120122

• Gas stripping has also been developed as a cost-effective means to remove butanol and reduce any product inhibition.123

• At the molecular level, the high product yields with hyperproducing strains can be ascribed to a defective glucose transport system exhibiting poor regu­lation and a more efficient use of glucose during the solventogenic stage.124

• The demonstration that the ABE fermentation can utilize corn fiber sugars (glucose, xylose, arabinose, and galactose) and is not inhibited by major sugar degradation products of pretreated lignocellulosic substrates.125126

• Overexpression of a single clostridial gene to increase both solvent produc­tion and producer cell tolerance of product accumulation.127

• Improved understanding of the molecular events causing loss of productiv­ity in solventogenic strains spontaneously or during repeated subculturing or continuous fermentation.128129

A technoeconomic evaluation of a production facility with an annual capacity of 153,000 tonnes published in 2001 estimated production costs for butanol of $0.29/kg ($0.24/l, assuming a density of 0.8098 kg/l, or $0.89/gallon), assuming a conversion
efficiency of 0.50 g products per gram of glucose and corn as the feedstock.130 The calculations were noted to be very sensitive to the price paid for the corn, the worst — case scenario costs reaching $1.07/kg; with the best-case scenario, the production costs were probably competitive with conventional gasoline (at that time showing a high degree of price instability), allowing for a lower energy content (figure 5.1).

The downstream processing operations for the ABE fermentation are necessar­ily more complex than for fermentations with single product, for example, ethanol. Not only can the insoluble materials from the harvested fermentation be used as a source for animal feed production, but the fermentation broth must be efficiently fractionated to maximize the economic returns possible from three saleable solvent products. Detailed analysis of a conventional downstream process modeled solvent extraction (by 2-ethyl-1-hexanol), solvent stripping, and two distillation steps to recover 96% of the butanol from a butanol-dominated mix of products.131 An optimal arrangement of these downstream steps could reduce the operating costs by 22%.

Advanced bioprocess options have included the following:

• A continuous two-stage fermentation design to maintain the producing cells in the solventogenic stage132

• Packed bed biofilm reactors with C. acetobutylicum and C. beijerinckii133

• A continuous production system with a high cell density obtained by cell recycling and capable of operation for more than 200 hours without strain degeneration or loss of productivity134

• Simultaneous saccharification and fermentation processes have been inves­tigated by adding exogenous cellulase to poorly cellulolytic strains135

A novel feedstock for biobutanol production is sludge, that is, the waste product in activated sludge processes for wastewater treatments; this material is generated at 4 x 107 m3/year in Japan and most is discharged by dumping.136 Adding glucose to the sludge supported growth and butanol production and a marked reduction in the content of suspended solids within 24 hours. In the Netherlands, domestic organic waste, that is, food residues, have been tested as substrates for the clostridial ABE fermentation, using chemical and enzymic pretreatments; growth and ABE forma­tion were supported mainly by soluble sugars, and steam pretreatment produced inhibitors of either growth or solvent formation.137138

Echoing the theme of recycling is the MixAlco process, developed at Texas A&M University, College Station, Texas; this can accept sewage and industrial sludges, manure, agricultural residues, or sorted municipal waste) as a feedstock, treated with lime and mixed with acid-forming organism from a saline environment to produce a mixture of alcohols that are subsequently thermally converted to ketones and hydro­genated to alcohols, predominately propanol but including higher alcohols.139 This is another fermentation technology awaiting testing at a practical commercial scale.

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.

A key change in the pricing structure of industrial alcohol in the United States occurred in the decade after 1975: the price of petrochemical ethylene showed an increase of nearly tenfold, and this steep rise in feedstock costs pushed the price of synthetic industrial alcohol from 150/l (570/gallon) to 530/l ($2.01/gallon).16 Corn prices fell significantly (from $129/ton to $87/ton) between 1984 and early 1988; because the coproduct costs were increasing as a percentage of the corn feedstock cost at that time, the “net corn cost” for ethanol production (the net cost as delivered to the ethanol production plant minus the revenue obtained by selling the coprod­ucts) and the net corn cost per unit volume of ethanol were both halved (figure 5.3).

By 1988, the costs involved in corn-derived ethanol production were entirely competitive with those of synthetic industrial alcohol (table 5.5). The major concern was that unexpectedly high investment costs could place a great strain on the eco­nomics of the process if the selling price for ethanol dipped: in general, such costs could be minimized by adding on anhydrous ethanol capacity to an existing bever­age alcohol plant or adding an ethanol production process to a starch or corn syrup plant, but expensive grassroots projects could face financial problems. Across the whole range of production facilities (small and large, new or with added capacity,

and with varying investment burdens), a manufacturing price for ethanol could be as low as 180/l (680/gallon) or as high as 420/l ($1.59/gallon).16 By 1988, the average fuel ethanol selling price had fallen below 3O0/l ($1.14/gallon), an economic move­ment that would have placed severe pressures on farm-scale production business plans (see section 5.2.1.4). As an incentive to fuel ethanol production and continuing the developments noted above (section 5.2.1.2), federal excise tax concession of 160/l (150/gallon), discounting by individual states by as much as 210/l (610/gallon), and direct payments by states to producers amounting to as much as 110/l (420/gallon), in conjunction with loan guarantees and urban development grants, encouraged the development of production by grassroots initiatives.16 Industrial-size facilities, built without special incentives, were already reaching capacities higher than 1 billion gallons/year as large corporations began to realize the earning potential of fuel etha­nol in what might become a consumer-led and consumer-oriented market.

It is highly doubtful that industrial biohydrogen processes will be the entry points for the widespread use of H2 as a fuel. Despite a number of major national and inter­national initiatives and research programs, fossil fuel-based and alternative energy processes are widely considered to be essential before 2030, or even as late as 2050. Of these nonbiological technologies, H2 production by coal gasification is clearly the worst alternative in terms of fossil energy use and greenhouse gas emissions (figure 7.8).93 Nevertheless, gasification and electricity-powered electrolytic routes to H2 offer the promise of production costs rivaling or even less than those of conventional gasoline for use in fuel cell-powered vehicles with an anticipated fuel economy approximately twice that of conventional internal combustion engines (figure 7.9). As a carbonless production route, the internationally accepted “route map” is the sulfur-iodine cycle based on the three reactions:

H2SO4 ^ SO2 + H2O + /O2 [850°C]

I2 + SO2 + 2H2O ^ 2HI + H2SO4 [120°C]

2HI ^ H2 + I2 [220-330°C]

The high temperatures required for the first reaction have prompted research pro­grams investigating solar-furnace splitting of sulfuric acid, for example, in the five — nation project HYTHEC (HYdrogen THErmochemical Cycles), involving research teams from France, Germany, Spain, Italy, and the United Kingdom in the “search for a long-term massive hydrogen production route” that would be sustainable and independent of fossil fuel reserves (www. hythec. org).

Electrolysis (wind)

The enormous added bonus of biohydrogen would be the use of other highly renewable resources as well as avoiding undue reliance on nuclear technology (an alternative means of providing the power for very-high-temperature reactors), highly persuasive rationales for the continuing interest in biohydrogen energy in the twenty-first century as exemplified by the International Energy Agency’s Hydrogen Implementing Agreement whose Task 15 involves Canada, Japan, Norway, Sweden, the Netherlands, the United Kingdom, and the United States in four R&D areas:94

• Light-driven H2 production by microalgae

• Maximizing photosynthetic efficiencies

• H2 fermentations

• Improving photobioreactors for H2 production

In Japan, all the major automobile manufacturers are active in the development of fuel cell-powered vehicles: Toyota, Honda, Nissan, Mazda, Daihatsu, Mitsubishi, and Suzuki.94

In Europe, HYVOLUTION is a program with partners from 11 European Union countries, Russia, and Turkey, funded by approximately $9.5 million, and aiming to establish decentralized H2 production from biomass, maximize the number and diversity of H2 production routes, and increase energy security of supply at both local and regional levels (www. biohydrogen. nl/hyvolution). The approach is based on combined bioprocesses with thermophilic and phototrophic bacteria to provide H2
production with high efficiencies in small-scale, cost-effective industries to reduce H2 production costs to $10/GJ by 2020 — with production costs in the $5-7/GJ range, biomass-derived H2 would be highly competitive with conventional fuels or biofuels.95 Principal subobjectives for HYVOLUTION include the following:

• Pretreatment technologies to optimize biodegradation of energy crops

• Maximized conversion of biomass to H2

• Assessment of installations for optimal gas cleaning

• Minimum energy demand and maximal product output

• Identification of market opportunities for a broad feedstock range

Based in Sweden, the SOLAR H program links molecular genetics and biomimetic chemistry to explore radically innovative approaches to renewable H2 production, including artificial photosynthesis in manmade systems (www. fotmol. uu. se). Japanese research has already explored aspects of this interface between industrial chemistry and photobiology, for example, incorporating an artificial chlorophyll (with a zinc ion replacing the green plant choice of magnesium) in a laboratory system with sucrose, the enzymes invertase and glucose oxidase, together with a platinum colloid to photoevolve H2.96

The size of the investment required to bring the hydrogen economy to fruition remains, however, daunting: from several billion to a few trillion dollars for several decades.97 The International Energy Agency also estimates that H2 production costs must be reduced by three — to tenfold and fuel cell costs by ten — to fiftyfold. Stationary fuel cells could represent 2-3% of global generating capacity by 2050, and total H2 use could reach 15.7 EJ by then. There are some appreciated risks in these prognos­tications, with governments holding back from imposing fuel taxes on H2 but impos­ing high CO2 penalties being strongly positive for increasing the possible use of H2, whereas high fuel cell prices for automobiles will be equally negative (figure 7.10).

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