Biodiesel, Fischer-Tropsch Diesel, and "Bio-ols"

6.1 BIODIESEL: CHEMISTRY AND PRODUCTION PROCESSES

6.1.1 Vegetable Oils and Chemically Processed Biofuels

Practical interest in the oils extracted from plant seeds as sources of usable transportation fuels has a historical lineage back to Rudolf Diesel and Henry Ford. Minimally refined vegetable oils can be blended with conventional diesel fuels and, if a 10% lower energy content of widely available oils (on a volume basis) is accept­able — with the consequent reduction of maximum fuel energy but without modifi­cation of the injection system — diesel fuel extenders are cheap and plentiful.1 There is much anecdotal evidence for diesel tanks being illegally “topped up” with vegeta­ble oils, reducing fuel costs but risking detection via the unusual aromas emanating from the tail pipe.

Industrial production has, however, focused on transforming vegetable oils into a mixture of fatty acid esters by a process frequently described as being akin to the “cracking” of petroleum, that is, a transesterification of triglycerides with low-molecular-weight alcohols (figure 6.1). This produces a higher-volatility mix­ture (“biodiesel”) with physicochemical properties much more similar to those of conventional diesel fluids (table 6.1). Biodiesel is not, as such, a biotechnological product, being manufactured with any suitable vegetable oil from crops with no history of plant biotechnology (or even from animal fats) by an entirely chemical procedure but commentators include biodiesel in the portfolio of emerging biofuels because of its biological origin as a plant seed oil. In 2005, the estimated world production of biodiesel was 2.91 million tonnes of oil equivalent, of which 87% was manufactured in the European Union (62% in Germany), with only the United States (7.5%) and Brazil (1.7%) as other major producers; this total supply amounted to less than 20% of that of global fuel ethanol production.2 World biodiesel supply had, on the other hand, increased by threefold between 2000 and 2005, and a marked accel­eration in the United States as well as in Europe is predicted by the International Energy Agency up to 2030.

Soybean oil dominates U. S. biodiesel production in existing and planned production facilities designed for single-oil use, but more of the operating or planned sites are capable of handling multiple feedstocks, including animal fats, recycled cooking oils,

and vegetable oils (figure 6.2).3 Figures supplied by the National Diesel Board (www. nbb. org) on September 2007 indicate 165 sites then operational in the United States with a total national production capacity of 1.85 billion gallons/year, with a further 80 sites under construction, with a total capacity expansion of 1.37 billion gallons/ year — the range of sizes of the sites is enormous, with (at one extreme) a site capable of producing 100 million gallons, dwarfing the smallest site (annual capacity 50,000 gallons). Almost as striking is the great geographical spread of biodiesel producers, with only 4 of the lower 48 states not being represented by late 2007.[59]

In the European Union, rape (canola) is the most abundant suitable monoculture crop, with the particular advantage of being readily cultivated in the relatively cold climates of northern Europe.4 It is, however, the sheer variety of single or mixed sources of oil and fat that could be transformed into biodiesel that has attracted both large-scale and niche-market industrial interest — at one extreme, even used

cooking oil (manufactured initially from corn, sunflower, etc.) can serve as the bio­logical input, a widely publicized example of exemplary social recycling.5

It is not surprising that critics of biofuels are skeptical about (or hostile to) further research into bioproduction routes, especially from food crop sources, because the proponents of biofuels have often confounded two quite separate issues:

1. Plant biomass as a supply of feedstocks for fuels for automobiles owned and operated by private drivers

2. Plant biomass as a source of industrial chemicals to replace petrochemicals “eventually” or “sooner or later”

Biofuels need not be manufactured from food crops, but both biofuels and biore­fineries must utilize lignocellulosic substrates to satisfy the massive demand that is presently met worldwide by a very large manufacturing sector, that is, the oil, gas, and petrochemical industries, based on an unsustainable basis (fossil fuels).

In addition, biofuels may be a poor choice in the long term when compared with photovoltaic cells to capture solar energy and the plethora of “renewable” (wind, wave, geothermal, etc.) energy sources, alternatives that have many dedicated publicists.

Further scientific research from a biotechnological perspective is unlikely to stumble on arguments to convince the critics of biofuels to radically revise their position or dubious investors to reassess the risks. Based on the material presented in this and the preceding seven chapters, however, a priority list can be assembled for bioenergy aims that are achievable within the next three to four decades (chapter 5, section 5.6), that is, before oil depletion becomes acute — viewed not purely as the narrow biofuels agenda but as part of the unavoidable need to develop a viable biocommodity production system within that time.

First, biohydrogen is the sole means of breaking any dependence or “addiction” to CO2 cycles in the industrial world. Is either photoproduction or “dark” bacterial fer­mentation energetically sufficient and cost-efficient to seriously rival solar-powered or chemical routes to generate the enormous quantities required for fuel cell technol­ogies on a global scale? Critics of fuel cell-powered mass transportation point to the cost of the units and the practical problems inherent in supplying inflammable H2. In October 2007, however, a report appeared of an elegant solution to both issues: a fuel cell designed for automobiles without any expensive platinum catalyst and capable of using hydrazine (N2H4) as a convenient liquid form of H2.101 The production of hydrazine has a total energy efficiency of 79% and the refilling energy efficiency of hydrazine is higher than of H2 because energy is required to compress gaseous H2. Whether this direct hydrazine fuel cell proves to be the final form of a vehicle — compatible fuel cell, a similar design is likely to evolved within the next 20-50 years. A viable technology to bioproduce H2 will, therefore, be highly desirable on an industrial scale to eliminate reliance on natural gas as the major long-term source of H2 for this supposedly zero-carbon fuel.

Second, lignocellulosic biomass is the main biological source of fuel ethanol (and/or other biofuels) on a truly mass-sufficient basis to displace (or replace) gaso­line. Can bioprocesses achieve the volumes of product required to displace up to 30% of gasoline demand before 2030, with or without tax incentives, as a nascent biocommodity sector seeking funding, with multiple, rival microbial catalysts and possible substrates, and with significant governmental guidance (or interference) to ensure continual development despite any short-term fluctuations in oil price or availability? Lignocelluloses remain refractory — this is particularly obvious when compared with the ease of processing of sugarcane and corn grains — and biotech­nology might profitably be applied to optimizing the use of clostridial microbes that have evolved during a billion years or more precisely to utilize the food resource that cellulose presents. Clostridial cellulases are a much-undervalued source of novel biocatalysts.102,103 Fungal cellulases can, as a further innovation, be incorpo­rated into bacterial cellulosomic structures with enhanced activity toward cellulosic substrates.104 Clostridia can even perform the ultimate in resource-efficient biofuels production, transforming domestic organic waste to ethanol and butanol.105,106 As arguments rage over the use of food crops and agriculturally valuable land for bio­fuels production, exploiting what the global ecosphere offers in efficient microbial biocatalysts adept at recycling the mountains of waste produced by human com­munities remains not only environmentally attractive but could, as the twenty-first century progresses, make overwhelming economic sense.

Third — and crucial to the success of biofuels production and biorefineries — have the best sources of the optimal choice of carbohydrate polymer — and lignin­degrading enzymes been located? The identification, gene mining, characterization, and successful manufacture of complex mixtures of enzymes to pretreat efficiently, rapidly, and without generating any unwanted or toxic products and the full range of mechanically disintegrated biomass feedstocks that could be available (on a seasonal or serendipitous or opportunistic basis) are essential to the rise of the much-antici­pated biobased economy. Unconventional and little-researched microorganisms are still fertile grounds for exploring even as intensively investigated enzymes as cel — lulases but artificially designed, multiple enzyme mixtures still appear to mimic the natural microbial world in offering the highest bioprocessing capabilities.107

Fourth, lignins cannot be eliminated (as essential components of terrestrial plant structure), but their carbon is underused and undervalued. Can laccases and other enzymes or whole wood-rotting cells be developed to liberate the benzenoid struc­tures that are presently petrochemical products? Lignins can also be inputs for hydro — processed high-octane fuels.108 Could they be enzymically processed at sufficiently rapid rates and with high carbon recoveries to enter the list of biorefinery resources? Broadening the question of the likely feedstocks for the hypothetical future biorefiner­ies, can new procedures (based on enzymes or mild chemical treatments or a mixture of both) define better ways of separating and fractionating plant biomass constitu­ents, that is, are presently operated feedstock pretreatment techniques for bioethanol production (some of which have been known and discussed for decades) only crude approximations to what is required for fully developed biorefinery operations?109111

Fifth, optimizing the interface between biorefineries and “traditional” fermen­tation manufacture will smooth the transition to the biobased economy. Can exist­ing fermentation processes operated at commercial scales of production be adapted to at least comparable yields and economic cost with novel sources of ingredients for microbial media presented by refineries?112,113 The industrial fermentation sector evolved under conditions where the supply of inputs usually mirrored products for the food industry (glucose syrups, soybean oil, soybean protein, etc.). Locating the manufacture of fine chemicals alongside the primary processing facilities for biore­fineries allows economies of scale and shared use but will require the rethinking and reformulation of media, nutrient supply, and feeding strategies to efficiently utilize the sugar units and other plant-derived product streams that will be available on an increasingly large scale.

Finally, harmonizing agronomy and biotechnology will accelerate the approach to the biorefinery model of the future commodity chemicals market. Plant biotechnol­ogists view the woefully low efficiency of capture of solar energy as a prime target for genetic therapy, and this issue may still limit greatly how much of the world’s present lavish energy use could be met by biomass as the ultimate bioenergy source.114 At the same time, the debate over the ecological desirability of monoculture plantations as sustainably supplying biomass for biofuels production remains a pointed argument — grassland pastures may simply outperform any dedicated “energy crops.”115

In the prelude to the establishment of a biorefineries for advanced biofuels, com­modity chemicals, or both, however, there is still time to resolve an equally per­tinent conundrum: should reliable crop as biorefinery inputs be (quickly) defined, relevant processing technologies fixed, products and coproducts agreed (nationally, if not internationally), and the various interdependent operations of a biorefinery be designed around those agronomic choices?116,117 Or is the clearly most energy-effi­cient route of choice for biomass use that of thermochemical decomposition (domesti­cally and in localized industry via wood-burning stoves of high energy conversions)? Biomass gasification dovetails very well into the existing oil and natural gas sectors and their chemistries, and in the first half of the twenty-first century Fischer-Tropsch liquid fuels remain an obvious short-term choice. Syngas is highly versatile, but can expanding the knowledge base fully exploit this versatile carbon and energy source? In particular, can syngas-based fermentations be the optimum biomass conversion methodology for bioethanol production?118

DEVELOPMENT: STAGNATION OR CONSOLIDATION?

Iogen’s process in Canada and (insofar as process details can be assessed) from Spanish and other imminent facilities use only a small fraction of the technologies that have been devised for ethanol production from lignocellulosic sources; patents and patent applications covering the last 30 years are exemplified by those listed in table 4.6.

Can any predictions be made about the take-up of innovative process methodolo­gies in the first decade of commercial fuel ethanol production from biomass? Retrac­ing steps across the 10-12 years since the manuscripts were prepared for the 1996 Handbook on Bioethanol publication reveals a significant divergence:

• “Ethanol from lignocellulosic biomass (bioethanol) can now be produced at costs competitive with the market price of ethanol from corn.”320

• “Although bioethanol production is competitive now for blending with gas­oline, the goal of the Department of Energy Biofuels Program is to lower the cost of production to $0.18/l, which is competitive with the price of gasoline from petroleum at $25/bbl with no special tax considerations.”321

• “Despite high capital costs, Charles Wyman of the University of California, Riverside, US, told Chemistry World that cellulose technology could be commercialized now if investors would take the risk or government provide more policy and financial assistance. That breakthrough may happen in the next five years.”322

Despite crude oil prices being considerably higher than $25/bbl since 2002 (figure 1.3), and despite two successive Presidential State of the Union addresses outlining a consistent vision for alternative fuels in the United States, only tax breaks may accelerate cellulosic ethanol production in an uncertain market, or the equivalence between corn and cellulosic ethanol production costs has been broken, or the 1990s may have seen too many overly optimistic forward statements.

Or, the successes of corn and sugar ethanol confused rather than illuminated. Together with biodiesel (a product with a very straightforward technology platform,

TABLE 4.5

Patent Applications and Patents Awarded for Plant Genetic Applications Relevant to Bioethanol Production

Title

Transgenic plants expressing cellulolytic enzymes Expression of enzymes involved in cellulose modification Expression of enzymes involved in cellulose modification

Manipulation of the phenolic acid content and digestibility of
plant cell walls…

Regulation and manipulation of sucrose content in sugarcane
Self-processing plants and plant parts

Genetic engineering of plants through manipulation of lignin
biosynthesis

Manipulation of the phenolic acid content and digestibility of
plant cell walls…

Commercial production of polysaccharide degrading enzymes in
plants…

Modification of plant lignin content

Transgenic fiber producing plants with increased expression of
sucrose synthetase

Transgenic plants containing ligninase and cellulase…

TABLE 4.6

Patent Applications and Patents Awarded for Cellulosic Ethanol Technologies

Date of filing or award Title Applicant/assignee

February 22, 1977 Process for making ethanol from cellulosic material using plural Bio-Industries, Inc., Hialeah, FL

ferments

December 9, 1986 Method for the conversion of a P & W substrate to glucose using Parsons and Whittemore, Inc. (NY)

Microspora bispora strain Rutgers P & W

October 10, 2000 Ethanol production using a soy hydrolysate-based medium or a University of Florida, Gainesville, FL

yeast autolysate-based medium

Ethanol production from lignocellulose University of Florida, Gainesville, FL

Method of processing lignocellulosic feedstock for enhanced Iogen Bio-Products Corporation, Ontario,

xylose and ethanol production Canada

Method of treating lignocellulosic biomass to produce cellulose Pure Vision Technology, Inc., Fort Lupton, CO

Organic biomass fractionation process E. S. Prior

Method for processing lignocellulosic material Forskningscenter Riso, Roskilde, Denmark

Methods for cost-effective saccharification of lignocellulosic E. E. Hood and J. A. Howard

biomass

Ethanol production with dilute acid hydrolysis using partially dried Midwest Research Institute, Kansas City, MO lignocellulosics

Recombinant hosts suitable for simultaneous saccharification and University of Florida Research Foundation, Inc. fermentation

Methods to enhance the activity of lignocellulose-degrading Athenix Corporation, Durham, NC

enzymes

Process to extract phenolic compounds from a residual plant Centro de Investigaciones Energeticas, Madrid, material using a hydrothermal treatment Spain

Procedure for the production of ethanol from lignocellulosic
biomass using a new heat-tolerant yeast

Methods for degrading lignocellulosic materials
Methods for degrading or converting plant cell wall
polysaccharides

Methods for glucose production using endoglucanase core protein
for improved recovery and reuse of enzyme
Methods and compositions for simultaneous saccharification and
fermentation

Upflow settling reactor for enzymatic hydrolysis of cellulose
Pretreatment of bales of feedstock
Methods and processing lignocellulosic feedstock for enhanced
xylose and ethanol production

Centro de Investigaciones Energeticas US 2005/0069998 A1 Medioambientales у Tecnologicas, Madrid, Spain Novozymes Biotech, Inc., Davis, CA US 2005/0164355 A1 Novozymes Biotech, Inc., Davis, CA US 2005/0233423 A1
D. Wahnon et al.
US 2006/008885 Al
University of Florida Research Foundation, Inc. US 7026152
B. Foody et al. Iogen Energy Corporation, Ottawa, Canada R. Griffin et al.
US 2006/0154352 Al US 7198925 US 2007/0148751 Al

see chapter 6, section 6.1), conservative technologies proved both profitable and easily scaleable. Biodiesel in particular had the temptation of untapped resources of plant materials (oils) that could, with no time-consuming or costly processing, become a second-generation biofuel after alcohol from sugar and corn.323 Designs of cellulosic ethanol plants still face the crucial problem of choice, especially that of pretreatment technology (section 4.2) for any of the large-scale feedstocks presently under serious consideration — and there may be simply too many possible choices, all supported by published data sets. In addition, there is the question of timing: the marked acceleration and expansion of fuel ethanol production in the United States started in the late 1990s with corn ethanol and has continued with corn ethanol for at least eight years (figure 1.16). In other words, the actual expansion of the industry could have occurred (and did occur) without exhausting the possible supply of corn, the feedstock with, by far, the easiest substrate preparation route before fermentation that could use the existing corn milling infrastructure.

What now (2008) and in the near future? The almost exclusive focus on corn in North America has certainly caused price inflation that has translated to higher feed costs for beef, pork, and chicken that will (as USDA predicts) result in a declin­ing meat supply.324 Higher corn prices have also triggered a tortilla inflation crisis in Mexico, and another USDA prognosis is for a 3.5% annual rise in food prices in 2007. These economic changes will only be exacerbated by a continued expansion of corn ethanol outputs without a massive surge in corn grain yield; lignocellulosics break this vicious cycle, adding diversity to the limited range of substrates but inevi­tably requiring more complex bioprocess technologies — of which the Iogen and Abengoa initiatives represent add-on solutions to existing fuel alcohol production processes. On this analysis, the more advanced process options remain for another five to ten years, and possibly much longer, while investors follow the proven tech­nologies (wheat straw, separate hydrolysis and fermentation, batch or fed-batch fer­mentation), minimizing production costs by sharing facilities with cereal ethanol production: the first cellulosic ethanol facilities in the United States may be opera­tional in 2010, possibly utilizing sugarcane bagasse feedstock, whereas the same recombinant bacterial technologies will be producing 4 million liters of ethanol from demolition wood waste in Japan by 2008 (www. verenium. com).

The step change beyond mixed cereal/cellulosic ethanol has, however, already been extensively discussed, that of the integrated production of biofuel, power, animal feeds, and chemical coproducts to maximize the number of different saleable commodities to supplement or (if need be, under adverse market conditions) supplant bioethanol as the income stream, that is, “biocommodity engineering.”325 Given the availability of large amounts of biomass sources in probably overlapping and complementary forms (thus necessitating flexibility in the production process to utilize multiple substrates on a seasonal basis), biocommodities will become no more of a process and production chal­lenge than are the different alcoholic outputs from most large breweries or, with a chem­ical methodology, those from industrial petroleum “cracking” into petrochemicals.

Investors will nevertheless require convincing of the economic viability of bio­ethanol in the short term, in years where crop yields (corn, wheat, etc.) are better or worse than expected, when farm commodity prices fluctuate, and when interna­tional competition for the most widely sought biofuel feedstocks may have become significant and — most importantly — with or without continued “financial assis­tance” from national governments. The economics of bioethanol will be examined in the next chapter to define the extent of financial subsidies used or that still remain necessary and how high capital costs may inhibit the growth of the nascent industry.

7.1 BIODIESEL FROM MICROALGAE AND MICROBES

7.1.1 Marine and Aquatic Biotechnology

In mid-2007, a review in the journal Biotechnology Advances concluded that micro­algae appeared to be the only source of renewable biodiesel capable of meeting the global demand for petrodiesel transportation fuels.1 The main argument was that the oil productivity of selected microalgae greatly exceeds that of the best seed oil — producing terrestrial plants; although both life forms utilize sunlight as their ulti­mate energy source, microalgae do so far more efficiently than do crop plants. By then, three U. S. companies were developing commercial bioreactor technologies to produce biodiesels from “oilgae” (as the producing species have been termed): Greenfuel Technologies (Cambridge, Massachusetts), Solix Biofuels (Fort Collins, Colorado), and PetroSun (Scottsdale, Arkansas) via its subsidiary Algae Biofuels.

This is a very different scenario from that detailed in the 1998 close-out report on nearly decades of research funded by the U. S. DOE.2 This program had set out to investigate the production of biodiesel from high-lipid algae grown in ponds and utilizing waste CO2 from coal-fired power plants. The main achievements of the research were the following:

1. The establishment of a collection of 300 species (mostly green algae and diatoms), housed in Hawaii, that accumulated high levels of oils; some spe­cies were capable of growth under extreme conditions of temperature, pH, and salinity.

2. A much greater understanding of the physiology and biochemistry of intracellular oil accumulation — in particular, the complex relationships among nutrient starvation, cell growth rate, oil content, and overall oil productivity.

3. Significant advances in the molecular biology and genetics of algae,[62] including the first isolation from a photosynthetic organism of the gene encoding acetyl-CoA carboxylase, the first committed step in fatty acid biosynthesis.3

4. The development of large-surface-area (1000 m2) pond systems capable of utilization of 90% of the injected CO2.

Although algal production routes had the enormous advantage of not encroaching on arable land or other agricultural resources for food crops, the perceived problem in the 1998 report was the high cost of algal biodiesel relative to conventional automo­tive fuels, up to $69/barrel in 1996 prices; the higher the biological productivity, the lower the production costs, whereas using flue gas was more economical than buying CO2 supplies (figure 7.1). With crude oil prices then being $20/barrel or less, such production costs were disappointingly high (figure 1.3). Post 2000, oil prices three to four times higher allow a very different interpretation of the algal biodiesel option (figure 1.11).

The open-pond technology was not only the simplest but also the cheapest pro­duction choice. Closed-system production offered far more controllable growth envi­ronments for the algae, but the cost of even the simplest tubular photobioreactors were projected to have 10 times higher capital costs than open-pond designs. In addition, open-pond cultures had been commercialized for high-value algal chemical products— and any attempt at large-scale (>1 ton/year) closed-production systems had failed.2 Choices of location and species had dramatically increased productivity during the lifetime of the program, from 50 to 300 tonnes/hectare/year, close to the calculated theoretical maximum for solar energy conversion (10%). The report concluded, there­fore, that microalgal fuel production was not limited by engineering issues but by cultivation factors, including species control in large outdoor environments, harvest­ing methods, and overall lipid productivity. Encouragingly, the potential supply of industrial waste sources of CO2 in the United States by 2010 was estimated to be as high as 2.25 x 106 tonnes/year, with Fischer-Tropsch conversion plants from fossil

fuels (chapter 6, section 6.2) and gasification/combined cycle power facilities offering the largest amounts of CO2 at low cost prices (figure 7.2).

The era of high oil prices, interest in and research effort into algal sources of oils for biodiesel production has become more globally distributed. Typical of this recent change in scientific and technical priorities has been Chinese studies of Chlorella protothecoides — but with the cells grown heterotrophically[63] (using chemical nutrients) rather than photosynthetically with a source of CO2:

• Heterotrophically grown cells contained 57.9% oil, more than three times higher than in autotrophic (photosynthetically CO2-fixing) cells; chemical pyrolysis yielded an oil with a lower oxygen content, a higher heating value, a lower density, and a lower viscosity than autotrophic cell bio-oil.4

• With a corn powder hydrolysate as carbon source (rather than glucose), a high cell concentration could be achieved; the oil (55.2%) could be efficiently extracted with hexane as a solvent and converted to biodiesel by transesterification with an acid catalyst.5

• Optimization of the transesterification defined a temperature of 30°C and a methanol:oil molar ratio of 56:1, resulting in a process time of 4 hours.6

• The process could be upscaled from 5 to 11,000 l, maintaining the lipid content; hexane-extracted oil could be transformed to methyl esters using an immobilized lipase and with a transesterification efficiency of more than 98% within 12 hours.7

As a very fast-growing “crop” (in comparison with terrestrial species), microalgae can also be viewed (even if grown autotrophically in external environments) as a lignin-less biomass source, potentially capable of being used as the substrate for ethanol production as well as biodiesel.8

The year 1985 marked the end of the first decade of the national program to use sugarcane-derived ethanol as an import substitute for gasoline. Rival estimates of the cost of Brazilian fuel ethanol varied widely, from $35 to $90/barrel of gasoline replaced. Assessing the economic impact of the various subsidies available to alcohol producers was difficult but indicated a minimum unsubsidized price of $45/barrel on the same gasoline replacement basis.20 Assuming that gasoline was mostly manufac­tured from imported petroleum, the overall cost comparison between gasoline and nationally produced ethanol was close to achieving a balance, clearly so if the import surcharge then levied on imported oil was taken into consideration (table 5.8).

TABLE 5.8

Production Costs for Sugar-Derived Ethanol in Brazil by 1985

Cost

Ethanol

$10-12 per ton 65 liters per ton $0.09-0.11 per liter $0.264-0.295 per liter

1.2 liter ethanol per liter gasoline 1.0 liter ethanol per liter gasoline $50-65 per barrel gasoline replaced $42^7 per barrel gasoline replaced Gasoline $29 per barrel $2 per barrel $6 per barrel $10 per barrel $47 per barrel $41 per barrel

of the pentose[49] sugars (and all of the much smaller amount of hexoses) to generate more than 67 million liters (1.8 million gallons) of ethanol/year. The final production cost of 95% aqueous alcohol was equivalent to 480/l ($1.82/gallon) after allowance for financial costs and assuming a small net income from CO2 as a coproduct (table 5.11). An essential parameter was that of the extent to which the cells could be recycled: single-batch use of the cells increased the production cost to 640/l ($2.42/gallon). The single largest contributor to the production cost was, however, the financial burden of repaying the investment in the plant, that is, more than 37% of the total annual production cost outlay (table 5.11).

of transportation fuels as the market for fuel ethanol imposes competitive pricing; the second point is a natural conclusion from the vast efforts invested in developing recombinant producing organism and bioprocesses (chapters 3 and 4, respectively).

The Swedish authors (from Lund University) have continued to explore cost models for ethanol from lignocellulosic substrates:

• SSF bioprocesses offer improved economics over standard separate hydro­lysis and fermentation because of higher ethanol yields and reduced capital costs; with softwood biomass sources, there are also significant advantages if either process could be operated with higher levels of insoluble material and if recycling the stillage after distillation (“backsetting”), in principle reducing the production cost to $0.42/l, or $1.59/gallon (table 5.12).29

• Operating steam pretreatment of softwoods in two steps (at lower and higher temperatures) to maximize the recovery of hemicellulose sugars and cellulosic glucose, respectively, has a higher overall ethanol yield and reduced requirement of enzymes but is more capital-intensive and has a higher energy demand; the net result is no reduction in the production cost of ethanol (table 5.13); further improvement to the process, including a higher insoluble solids content for the second step, might reduce the pro­duction cost by 5-6%.30

The National Renewable Energy Laboratory, in association with consultant engi­neers, presented an outline cost model for the industrial-scale production of bioetha­nol (2000 tonnes/day consumption of feedstock, 52 million gallons of ethanol/year) from a hardwood yellow poplar biomass source.31 Operating costs were calculated to be approximately 620/gallon of ethanol (table 5.14). Discounted cash flow analysis assuming a discount rate of 10% indicated a minimum selling price of $1.44/gallon for a capital investment of $234 million. On the technical level, the key features of the envisaged process were

• Acid pretreatment of the biomass substrate (19% of the installed equip­ment cost)

• On-site generation of cellulase

• SSF of the pretreated substrate with a Zymomonas mobilis capable of utiliz­ing only glucose and xylose

• Wastewater treatment via anaerobic digestion to methane

• Utilization of three available waste fuel streams (methane, residual lignin solids, and a concentrated syrup from evaporation of the stillage) in a fluid­ized bed combustor, burner, and turbogenerator (33% of the installed equip­ment cost)

Although the complete array of technology in the model was unproven on a large scale, much of the process could be accurately described as “near term” or “based on the current status of research that is complete or nearly so.”31 The computed mini­mum selling price for ethanol was 20% higher than that of corn-derived ethanol, with a more than twofold greater investment cost ($4.50/gallon as compared with approximately $2/gallon).

TABLE 5.12

Cost Estimates for Ethanol Production from Softwood Using Different Bioprocess Technologies

Source: Data from Wingren et al.29

a Simultaneous saccharification and fermentation, 63 billion liters per year (base case) b Separate hydrolysis and fermentation, 55 billon liters per year (base case)

c 195,600 tonne raw material per year, operated continuously (8000 hours per year), notionally located in northern Sweden d C02 and solid fuel

TABLE 5.13

Cost Estimates for Ethanol Production from Softwood Using Different Pretreatment Options

Source: Data from Wingren et al.30

a 215oC, residence time 5 min, SO2 added to 2% of the water content of the wood; 47 billion liters etha­nol per year capacity

b 190oC, residence time 2 min, then 210oC for 5 min; SO2 added to 2% of the water content of the wood;

49 billion liters ethanol per year capacity c 200,000 tonnes per year; plant operating time 8000 hours per year d CO2 and solid fuel

Operating Costs for Yellow Poplar Sawdust-Ethanol in the United States

Source: Data from Wooley et al.31 a Poplar sawdust at $25 per tonne b Excess electricity sold to grid at 4 cents per kWh

TABLE 5.15

Estimated Production Costs for Bioethanol in 2003

Technical advances will define to what extent agricultural and wastewater resources can be transformed into quantities of electricity and liquid and gaseous fuels as energy carriers and to what extent these will contribute to local and national power demands, but certain industries could benefit greatly from considering their wastes as sources of bioenergy, either for immediate use or as a saleable commodity. To illustrate this, a bilateral study between Japan and Malaysia of the waste streams from the Malaysian palm oil industry (42.5 million tons in 2001) demonstrated that, with Enterobacter and Clostridium strains evolving H2 or producing acetone, buta­nol, and ethanol (chapter 6, section 6.3.3), more than 62,000 tons of oil equivalent in total energy could be generated.113

Biohydrogen (however it is produced by living cells) has the potential to mar­ginalize all other biofuels, from ethanol and biodiesel to all presently contemplated “bio” options for mass transportation — if, that is, onboard fuel cells achieve their cost and vehicle range targets. In one possible future, mariculture units growing cya­nobacteria in coastal waters will be the sources of renewable energy from water and sunlight, providing the H2 as the most environmentally friendly energy carrier.114 In another (Chinese) model, cultures of Propionibacterium, Clostridium, and Bacteri — odes species relentlessly ferment whatever substrates can be made available to them to form H2 and ethanol and/or mixtures of acids, quite independently of sunlight, and in processes that can be managed by simply adjusting the pH to determine the product stream.115

Even the hydrogen economy has its critics, however, and in another possible future, hybrid gasoline-electric vehicles will dominate the highways by as early as 2020, reducing gasoline and greenhouse gas emissions by 30-50%, with no major investments in fuel infrastructure; they may even be dually hybrid, being able to run on gasoline-biofuel blends, traveling 500 miles on a gallon of gasoline mixed with five gallons of cellulosic ethanol.116

When, in 1975, Ballard occupied a derelict motel in southern Arizona at the beginning of the quest to develop a viable technology to power an electric vehicle and so reduce dependency on fossil fuels, optimism may have been tempered with the realization that a long and uncertain journey had just commenced, but optimism was certainly rampant by the late 1990s.117 As an “entirely unauthorized” biography of Ballard (the early pacesetters in fuel cell technology for transportation) noted, the starting pistol for the race to develop a marketable fuel cell-powered automobile was fired in April 1997 when Daimler-Benz paid nearly $200 million for a 25% share in Ballard and committed itself to invest a further $300 million.118 A decade later, prog­ress continues worldwide, but projections about who crosses the winning line first and the eventual date of mass use of such ecofriendly vehicles tends to relentlessly slip back into “decades away” — inventors have, necessarily, to be ever hopeful about the futures of their brain children but underestimate production costs and the full sequence of engineering and other events that lead to commercialization.

Even if developments in the next two to three decades render both fuel ethanol and biodiesel obsolete, however, the many advances made in the biotechnology of the bioproduction of biofuels will not prove to be wasted. To return once more to the preface, biomass as the main supply of chemical feedstocks may be unavoidable in the twenty-first century as increased demand for gasoline in the rapidly developing economies of Asia and South America applies a price tourniquet to petrochemicals, particularly rapidly if oil reserves prove smaller than estimated or if an accelerat­ing CO2-dominated climate change forces political action to restrain CO2-producing industries.118 Can agricultural sources ever be justified as substrates for the produc­tion of transportation fuels? Or solely for automobile fuels?

Or can biofuels gain global approval as part of the mix of products emanating from biorefineries, in a flexible output that could replace petrochemicals, provide biofuels for blends according to market demands, and provide fuels for multiple types of fuel cells? The next and final chapter explores how the biorefinery concept emerged in the 1990s to be the beacon of a radically different vision of how biotech­nology and commodity chemical production can merge in another blueprint for a sustainable mobile and industrial society.

Biodiesel is unique among biofuels in not being a single, defined chemical compound but a variable mixture, even from a monoculture crop source. The triglycerides in any plant oil are a mixture of unsaturated and saturated fatty acids esterified to glycerol; fatty materials from land animals have much higher contents of saturated fatty acids (table 6.2).6 This variability has one far reaching implication: reducing the content of saturated fatty acid methyl esters in biodiesel reduces the cloud point, the tempera­ture below which crystallization becomes sufficiently advanced to plug fuel lines; a diesel suitable for winter use may have a cloud point below -11°C, and “winteriza­tion” (treatment at low temperature and removal of solidified material) of biodiesel generates a product with similar improved operability and startup characteristics.7,8

The idiosyncratic fatty acid content of canola seed oil, with its preponderance of the very long chain erucic acid (table 6.2), has a quite different significance. Erucic acid has been known since the 1950s to stimulate cholesterol synthesis by animals.9 The potential adverse health effects (increased risk of circulatory disease) led to leg­islation on the erucic acid content of edible oils and the development of low-erucic acid cultivars, whereas, by contrast, high-erucic acid oils have a market (estimated to be more than $120 million in 2004) because erucic acid and its derivatives are

Saturated Unsaturated

a Erucic (canola), C14 monoethenoic (soybean)

feedstocks for the manufacture of slip-promoting agents, surfactants, and other spe­cialized chemicals.10 High-erucic acid oils would be either desirable or neutral for biodiesel production, but low-erucic cultivars are higher yielding — and, in any case, legal requirements were in place in the European Union by 1992 to geographically separate the two types of “oilseed rape” cultivation to minimize cross-pollination and contamination of agricultural products intended for human consumption.4

The majority of the biodiesel producers continue to employ a base-catalyzed reaction with sodium or potassium hydroxide (figure 6.3).11 This has the economic attractions of low temperatures and pressures in the reaction, high conversion efficiencies in a single step, and no requirement for exotic materials in the construc­tion of the chemical reactor. The liberation of glycerol (sometimes referred to as “glycerine” or “glycerin”) in the transesterification reaction generates a potentially saleable coproduct (see section 6.3). The generation of fatty acid methyl esters is the same reaction as that to form volatile derivatives of fatty acids before their analysis by gas liquid chromatographic methods, and the key parameters for optimization are reaction time, temperature, and the molar ratio of oil to alcohol, but choices of

the type of catalyst used and the short-chain alcohol coreactant can also be made.12 Different oil types of plant origin have been the subject of process optimization studies; five recent examples, exemplifying the global nature of R&D activities with biodiesels, are summarized in table 6.3.13-17 More subtle factors include differential effects on product yield and purity; for example, temperature has a significant posi­tive effect on biodiesel purity but a negative influence on biodiesel yield, and the alcohol:oil molar ratio is only significant for biodiesel purity (with a positive influ­ence).17 Although the biodiesel yield increased at decreasing catalyst concentration and temperature, the methanol:oil ratio did not affect the material balance.18

A variety of novel catalysts have been explored, partly to avoid the use of caustic materials but also to facilitate catalyst recovery and reuse:

• Sulfonated amorphous carbon19

• Ion-exchange resins20

• Sodium ethoxide21

• Solid acid catalysts (e. g., ZnO)14

Indeed, the requirement for a catalyst can be eliminated if high temperatures and pressures are used to generate “supercritical” fluid conditions, under which alcohols can either react directly with triglycerides or (in two-stage procedures) with fatty acids liberated from triglycerides.22-24

Far greater attention has, however, been paid to developing a biotechnological approach to biodiesel production, employing enzyme catalysts, usually lipases, and employing their catalytic abilities to carry out transesterification (or alcohololysis) rather than straightforward hydrolyses of triglycerides to liberate free fatty acids and glycerol.25 The principal process advantage of the enzyme-based approach is the ability to use low to moderate temperatures and atmospheric pressure in the reaction vessel while ensuring little or no chemical decomposition (i. e., a high product purity); the main drawback is the much longer incubation times to achieve more than 90% conversion of the triglycerides, that is, up to 120 hours.26 The barrier to full commer­cialization is maintaining the (relatively expensive) enzyme active during repeated

batch use. Rival enzyme products show differing stability, and methanol appears to induce a faster loss of activity than does ethanol.27 Examples of enzyme-catalyzed processes using oils of plant origin and with prolonged survival of the lipases are summarized in table 6.4.26-29 Lipase from Pseudomonas cepacia was used in an immobilized form within a chemically inert, hydrophobic sol-gel support; under optimal conditions with soybean oil, high methyl and ethyl ester formations were achieved within a 1-hr reaction, and the immobilized lipase was consistently more active than the free enzyme, losing little activity when subjected to repeated uses.30 Stepwise addition of methanol to delay inactivation of the enzyme is another pos­sible strategy.31 Reversal of methanol-mediated inactivation of immobilized lipase has been demonstrated with higher alcohols (secondary and tertiary butanols).32 In the long term, molecular evolution technologies will develop lipases with reduced sensitivity to methanol and increased specific activities; in the short term, whole-cell biocatalysis has obvious potential for industrial application, offering on-site genera­tion of lipase activity in cell lines that could be selected to be robust for oil trans — esterification.33-35 As with cellulases (chapter 2, section 2.4.3), investigation of newly discovered microbes or extremophiles may reveal enzymes with properties particu­larly well suited for industrial use. A lipase-producing bacterium strain screened from soil samples of China, identified as Pseudomonas fluorescens, contains a novel psychrophilic lipase (with a temperature optimum of only 20°C); this may represent a highly competitive energy-saving biocatalyst because lipase-mediated biodiesel production is normally carried out at 35-50°C.36

Commercially available lipases and lipases identified in a wider spectrum of microbial enzyme producers can efficiently use different low-molecular-weight alcohols as substrates for transesterification. Substituting higher alcohols for metha­nol can maintain active lipase for much longer periods of continuous batch opera — tion.37 A research group in Italy has also exploited this lax substrate specificity to produce fatty acid esters from a mixture of linear and branched short-chain alcohols

that mimics the residual fusel oil left after ethanol; not only can this utilize a waste product from bioethanol production but the fatty acid esters are potentially impor­tant for biodiesels because they improve low-temperature properties.38

Crude oils can give poor transesterification rates because of their contents of free fatty acids and other components lost during refining; the free fatty acids (up to 3% of the oils) react with the alkaline catalysts and form saponified products during the transesterification. Crude soybean oil could be converted into methyl esters as well as refined oil if a lipase process was used, although a lengthy incubation period was again required.39 With an Indonesian seed oil (from Jatropha curcas) exhibiting very high free fatty acids (15%), a two-step pretreatment process was devised: the first step was carried out with sulfuric acid as catalyst in a reaction at 50°C and removing the methanol-water layer; the second step was a conventional transesterification using an alkaline catalyst to produce biodiesel at 65°C.40 Rice bran stored at room tempera­ture can show extensive (>75%) hydrolysis of triglycerides to free fatty acids; the suc­cessful processing of the oil fraction also required a two-step methanolysis process (but both steps being acid-catalyzed), resulting in a 98% methyl ester formation in less than 8 hr, and the coproduction of residue with high contents of nutraceuticals such as y-oryzanol and phytosterols.41 Supercritical methanol treatment (without any catalyst) at 350°C can generate esters from both triglycerides and free fatty acids, thus giving a simpler process with a higher total yield of biodeisel.42

Other innovations in biodiesel production have included the following:

• A six-stage continuous reactor for transesterification of palm oil in Thai­land, claimed to produce saleable biodiesel within residence time of six minutes in a laboratory prototype with a production capacity of 17.3 l/hr43

• A Romanian bench-scale continuous process for the manufacture of biodiesel from crude vegetable oils under high-power, low-frequency ultrasonic irradiation44

• A two-phase membrane reactor developed to produce biodiesel from canola oil and methanol (this combination is immiscible, providing a mass-trans­fer challenge in the early stages of the transesterification); this Canadian design of reactor is particularly useful in removing unreacted oil from the product, yielding high-purity biodiesel and shifting the reaction equilib­rium to the product side45

• A novel enzyme-catalyzed biodiesel process was developed to avoid the liberation of glycerol from triglycerides, maximizing the carbon recovery in the product; methyl acetate replaced methanol, and the resulting triacetylglycerol had no negative effect on the fuel properties of the biodiesel46

After biodiesel production, the fuel’s thermal properties have been improved — in this case, to reduce the onset of volatilization (table 6.1) of soybean-derived biodiesel to below that of conventional diesel — by ozonolysis; the onset freezing temperature of ozonated methyl soyate was reduced from -63°C to -86°C.47

The most radical development in biodiesel production has, however, been in Brazil where PETROBRAS has combined mineral and biological oils in the H-BIO
process; few details have been made public, but the essential step is to add a vegetable oil to the straight-run diesel, gasoil, and coker gasoil fractions from the refining pro­cess, the total streams then being catalytically hydrogenated.48 The triglycerides are transformed into linear hydrocarbon chains, similar to the hydrocarbons in the petro­leum oil streams; the conversion of triglycerides is high (at least 95%), with a small propane coproduct. Because the process takes advantage of the existing infrastruc­ture of an oil refinery, the potential exists for an orderly transition from conventional diesel to biodiesel blends, with a gradual increase in the “bio” input if (as widely predicted) oil reserves dwindle (chapter 5, section 5.6).

the Petrochemical Industry?

There is little doubt that biotechnology has — or will — produce viable solutions to all the manifold problems inherent in processing lignocellulosic biomass with maximum efficiency into ethanol and other advanced biofuels. But are these tech­niques and methodologies simply too elaborate and overtechnologized for the task in hand? Although academic groups insert increasingly complex and inclusive arrays of genes into microorganisms to metabolize the whole gamut of sugars pres­ent in woody feedstocks, is this the realistic technology toolbox for the massive amounts of primary chemical building blocks and biofuels that will (eventually) be required?

Of the three established economically significant biofuels presently produced, two (starch — and sugar-derived ethanol) have required only modest advances in carbohy­drate-processing enzymes as essential inputs from industrial biotechnology, whereas the third (biodiesel) has evolved as an entirely chemical operation. Recent applica­tions of chemical catalysis to the production of fuels and other value-added chemicals from biomass-derived oxygenated feedstocks have now opened up routes involving well-defined reactions of organic chemistry (dehydration/dehydrogenation, aldol con­densation, etc.).119 To the process chemist’s eyes, biorefineries are entirely analogous to petrochemical refineries in that a limited range of major feedstocks — biomass hydrolysates, vegetable oils, pyrolytic bio-oils, lignin, and others (in biorefineries) and oils and diesel fuel (in a petrochemical refinery) — are convertible by cata­lytic cracking, hydrotreating, and hydrocracking to a range of fuels (gasoline, diesel, aviation fuel, and liquid petroleum gas) and chemicals (olefins, etc.).120 The logical extrapolation is that biomass-derived and fossil fuel inputs to refineries could be mixed — and trials of that concept have indeed already begun, with oil compa­nies exploring cofeeding of biomass and petroleum feedstocks, the production of biofuels in petroleum refineries, and the direct production of diesel fuel from veg­etable oils by a hydrotreatment process. Eventually (and perhaps within the next 20-60 years), the massive infrastructure of oil refineries may find their conventional feedstocks dwindling; the massive accumulated experience of chemical engineer­ing could easily accommodate new feedstock types and modified chemical catalytic processes. Although biotechnologists have focused for the past decade on refining ethanologens and other biofuels producers to utilize more of the available carbon in highly processed lignocellulosic substrates, the chemists can offer pyrolytic and thermochemical technologies that can transform all the available carbon in crude biomass to forms that can either be catalytically modified to value-added chemicals or act as inputs to Fischer-Tropsch fuel production streams. At high temperatures and pressures, given well-designed catalysts and predictable downstream operations, no material is “refractory.” If the investment in the chemical hardware has already been made, why not ease into a new feedstock regime in a form of “reeducation,” gradu­ally restructuring the industry to a sustainable future?

Between 20 and 60 years: either limit is allowed by present versions of the “Hubbert curve” before the question of how to replace dwindling oil reserves in the production of the myriad chemicals on which Western industrialized societies rely becomes acute.121 What biotechnology now makes very clear is that understanding the metabolism of even as ancient a “domesticated” microbe as the common etha — nologenic yeast cannot be achieved purely in terms of genes and gene function but must include fermentation media and operating engineering in any attempt to map the biochemistry and molecular biology accompanying ethanol formation and accu­mulation in real-world and real-time fermentors.122,123

That the two final scientific references in this volume explore the computational re-creation of metabolism inside the fungus whose secretion of cotton-destroying enzymes initiated modern scientific exploration of cellulases and in ethanol-forming yeast cells is entirely appropriate. Nine millennia have passed since ethanol-forming microbes were first consciously — and (possibly) wisely — used by Homo sapiens; in 2007, we learned how to construct computer simulations of yeast metabolic networks and perform dynamic analyses and were able to conclude that genetic information alone is unable to optimize any biochemical pathway or maximize the yield of etha­nol or any other fermentation product. Mathematical modeling, genetic manipulations, and the application of advanced chemical engineering can combine to achieve optima but only on the clear understanding that any conclusion may not be portable to simple change in circumstances (with glucose and xylose as cosubstrates rather than when individually consumed) or to each and every running of a complex series of events, that is, the biologically variable growth and metabolism of a microorganism under multiple physiological stresses in concentrated media and driven to maximal productivity.

Twenty, 40, 50, or 100 years: yeast cells (or at least, those in laboratory strains) have had little more than 20 years of deliberate, goal-driven molecular evolution, and this despite an incomplete understanding of gene expression and its endogenous regulation. “Scientific” studies of beer and wine yeasts, their inheritance patterns, stability, and metabolic capabilities may extend to a century. Pasteur was defining modern concepts of fermentation little more 150 years ago. The present generation of research scientists may be unique: it is the first to work with the sense of a severe time limit, not racing to launch a product or file a patent, but with a deadline, the realization that — however imprecisely computed is that future date — novel tech­nologies must be in place before petrochemicals become increasingly scarce and all ready-at-hand energy sources become valuable commodities to be bartered, in other words, an ideal time in which to put to good use all the knowledge gained over nine millennia of evolving biotechnological craft and science.

[1] Chinese texts from ca. 1000 BC warn against overindulgence in distilled spirits.

• Whisky (or whiskey) was widely known in Ireland by the time of the Nor­man invasion of 1170-1172.

• Arnold de Villeneuve, a French chemist, wrote the first treatise on distilla­tion, ca. 1310.

• A comprehensive text on distilling was published in Frankfurt-am-Main (Germany) in 1556.

• The production of brandies by the distillation of grape wines became widespread in France in the seventeenth century.

• The first recorded production of grain spirits in North America was that by the director general of the colony of New Netherland in 1640 (on Staten Island).

• In 1779, 1,152 stills had been registered in Ireland — this number had fallen drastically to 246 by 1790 as illicit “moonshine” pot stills flourished.

• In 1826, a continuously operating still was patented by Robert Stein of Clackmannanshire, Scotland.

• The twin-column distillation apparatus devised by the Irishman Aeneas Coffey was accepted by the Bureau of Excise of the United Kingdom in 1830; this apparatus, with many variations and improvements to the basic design, continues to yield high-proof ethanol (94-96% by volume).

[2] The Automobile Club of America sponsored a competition for alcohol-powered vehicles in 1906.

[3] High demand for oil exceeded predictions in 1970.

• The continued closure of the Suez Canal after the 1967 war between Israel and Egypt was confounded by a shortage of tanker tonnage for the much longer voyage around South Africa.

[4] In 1990, the Instituto do Agucar e do Alcool, the body through which govern­mental policy for ethanol production had been exercised, was abolished.

• In 1993, a law was passed that all gasoline sold in Brazil would have a minimum of 20% ethanol by volume.

[5] In 2004-2005, Brazil was the world’s largest producer of ethanol, with 37% of the total, that is, 4.5 billion gallons.

• Brazil exported 15% of its total ethanol production in 2005.

[6] The 1970 Clean Air Act (amended in 1977 and 1990) began the requirement for cleaner burning gasoline and (eventually) the mandatory inclusion of “oxygenates,” that is, oxygen-rich additives.

• The 1988 Alternative Motor Fuels Act promoted the development of ethanol and other alternative fuels and alternative-fuel vehicles (AFVs).

• The 1992 Energy Policy Act defined a broad range of alternative fuels but, more urgently, required that the federal vehicle fleet include an increasing number of AFVs and that they be powered by domestically produced alternative fuels.

[7] Six Canadian companies, starting operations between 1981 and 2006, with total capacity of 418 million liters of ethanol per year, mostly from wheat starch, with a further 390 million liters under construction (Canadian Renewable Fuels Strategy, Canadian Renewable Fuels Association, Toronto, Ontario, Canada, April 2006).

[8] Climatic factors including seasonal hours of sunlight, precipitation, average temperature, and others cer­tainly affect crop yield and will, therefore, impact on the economics of ethanol production as the cost of, for example, corn grain as an essential input into the ethanol production process varies from year to year.

0

oxygenated hydrocarbons. It is only at temperatures above 1100°C that the thermal conversion steps are efficient and only CO is a significant waste product of incomplete combustion (although even this can be minimized above a threshold residence time in the zone of highest temperatures); below 700°C, the use of catalytic conversion is essential to avoid serious air pollution with gases and volatilized material including tars.2 With catalytic combustors in residential wood burners designed to high engineering standards, overall efficiencies can be 80% of the theoretical maximum and particulate emissions as low as 1 g/kg.

Direct combustion is, however, only one of three thermochemical options for biomass utilization. Gasification (incomplete combustion) yields different mixes of products depending on the conditions used:2,3

• With pure oxygen as the combustant, a producer (or synthesis) gas with a high CO content is produced.

• The use of air rather than oxygen reduces the heating value because nitrogen dilutes the mixture of gases.

[10] If water is present and high temperatures are reached, hydrogen may also be formed, but excess water tends to result in high CO2 concentrations and greatly reduces the heating value of the gaseous product.

Producer/synthesis gas resulting from gasification technologies generally has a low heating value (4-10 MJ/L) and is best suited to in situ power heat and/ or generation. The third thermochemical method, pyrolysis (i. e., heating in the absence of air or oxygen) can be an efficient means of generating a gas high in hydrogen and CO but can also yield charcoal, a material with many (sometimes ancient) industrial uses.

[11] Ethanol is formed if a culture growing below the crucial rate of glucose entry is transferred to anaero­bic conditions; the faster the subcritical rate of feeding, the faster ethanol is formed after transfer to a “fermentation” environment.12

[12] Steam explosion was originally patented for fiberboard production in 1926. In the 1970s, batch and continuous processes were developed by the Iotech Corporation and the Stake Corporation, respec­tively, in Canada.

TABLE 2.2

Chemical Pretreatment Methods for Lignocellulosic Biomass

Method Principle Pilot plant use? a

Acids Hemicellulose solubilization

dilute sulfuric —

dilute hydrochloric —

dilute nitric —

dilute phosphoric —

steaming with sulfuric acid +

impregnation

steam explosion/sulfuric acid —

impregnation

steam explosion/sulfur dioxide —

steam explosion/carbon dioxide —

Alkalis Delignifi cation + hemicellulose

removal

sodium hydroxide —

sodium hydroxide + peroxide —

steam explosion/sodium —

hydroxide

aqueous ammonia —

calcium hydroxide —

Solvents Delignifi cation

methanol —

ethanol —

acetone —

a Data from Hsu.19

[13] The conversion of renewable substrates into single-cell protein was the topic in the 1960s and 1970s that most clearly resembles bioethanol production 30-40 years later (see, for example, reference 23). Historically it was the first major failure of industrial biotechnology.

[14] Historically, this organism (designated until recently as Trichoderma reesei) represents the beginning of biotechnological interest in cellulase, as it caused the U. S. Army major equipment and supply prob­lems in the Pacific during World War II by digesting military cotton garments.

[15] The susceptibility of sophorose to P-glucoside-catalyzed degradation precludes the possibility of the disaccharide being used as a recoverable “catalyst” for cellulase expression, although a chemical analog lacking the glycosidic linkage may be more stable and recyclable.

[16] Terrestrial and marine bacteria

• Yeasts and fungi

• Rumen bacteria and protozoa

• Marine algae

• Wood-digesting insects

• Molluscs and crustaceans

• Higher plants (in particular, in germinating seeds)

[17] Much of the report was focused on projections for meeting a much larger biomass supply by combination of altered land use and increased productivity. These topics are covered later, in chapter 5, section 5.4.

[18] cerevisiae is, from the standpoint of classical microbial physiology, best

described as “facultatively fermentative.” That is, it can metabolize sugars such as

glucose either entirely to CO2 and water given an adequate O2 supply or (under micro­

aerobic conditions) generate large amounts of ethanol; this ability for dual metabolism

is exhibited by a large number of yeast species.10 In complete anaerobiosis, however,

[23] There is nothing archaic about this area of microbial physiology, one of the effects being emphasized in the title of a 2002 patent (Production of Lactate Using Crabtree Negative Organisms in Varying Culture Conditions, U. S. Patent 6,485,947, November 26, 2002) awarded to Cargill Dow Polymers, LLC, Minnetonka, MN.

[24] So similar are P. stipitis and C. shehatae that they were at one time described as anamorphs of each other but the application of modern nucleic acid analytical techniques established that they are distinct biological species; from their ribosomal RNA sequences, C. shehatae has only recently (in biological time) diverged from the Pichia group, but both are well seperated from S. cerevisiae.57

[25] This fermentative route is not available to a strictly aerobic yeast such as Schizosaccharomyces pombe, which contains a mitochondrial DHOdehase and requires a fully functional mitochondrial electron transport chain for DHOdehase activity.137

[26] Redox balance is achieved because ethanol (oxidation value -2) and CO2 (oxidation value +2) effec­tively cancel one another out, lactate and acetate having zero values, thus leaving a small inaccuracy for the trace of succinate (oxidation value +1) formed in the slightly “leaky” mutant.

[27] Tropical counterparts of yogurts and fermented milk drinks prepared using Z. mobilis were known to the Aztecs for their therapeutic properties.194

[28] Symport is the simultaneous transport of a substrate and a cation (Na+, H+, etc.) in the same direction, while antiport is the exchange of two like-charged compounds (for example, Na+ and H+) via a common carrier; 40% of substrate uptake into bacterial cells requires one or the other of these two types of ion-driven transport.154

[29] High conversion yield

• High ethanol tolerance

• Tolerance to hydrolysates

• No O2 requirement (i. e., a facultative anaerobe)

[30] The assignee of an early U. S. patent for a microbe (a Kluyveromyces yeast) capable of producing alcohol from xylose and cellobiose (table 3.6).

[31] Acid prehydrolysis times had been described as “less than 1 minute” in 1999 but a more flexible regime had been instituted after the facility became operational (figure 4.1).

[32] Including Bacillus species then being developed by Agrol Ltd. (later, Agrol Biotechnologies Ltd.), a spinout company from the University of London, England.

[33] Denmark is entirely above latitude 54°N; the southernmost regions of Canada reach below 50°N; both are too far north to grow biomass crops other than some cereal species and forest tress (and Denmark has a limited land availability for large-scale tree plantations).

• Liquid flow-through enhances hemicellulose sugar yields, increases cel­lulose digestibility to enzyme treatment, and reduces unwanted chemi­cal reactions but with the associated penalties of high water and energy use; some of the benefits of flow-through can be achieved by limited fluid movement and exchange early in the acid digestion process.42-48’56-57

• Pretreated corn stovers appear to be much less toxic to ethanologens than other agricultural substrates.44- 4647

• Moreover — removal of acetic acid (a degradation product acetylated hemi­cellulose sugars) has been demonstrated at 25-35°C using activated carbon powder — and a natural fungus has been identified to metabolize furans and actively grow in dilute acid hydrolysates from corn stover.52-53

[35] This Chinese work is particularly relevant for improving the economics of bioethanol production because sugarcane bagasse represents the most abundant lignocellulosic agricultural material in southern China.97

[36] It is sometimes asserted that brewers disliked the increased emphasis on hygiene and “scientific” precision but offering such precise, reproducible technology was a positive and populat selling point in the 1970s, explicitly stated in advertisements for major brands of nationally sold beers and lagers in the United Kingdom and elsewhere.

[37] Utilizing the cell recycling principle developed in New Zealand but adding a vacuum-enhanced continuous recovery of ethanol from the fermentation

[38] Avoiding high temperature “excursions” in the stage of ethanol produc­tion — glycerol formation becomes uncoupled to growth and glycerol

[39] A continuously productive immobilized system with cane sugar molasses as the substrate proved to be stable for up to 60 days at a wide range of dilution rates, 0.05-3.00/hr.

• The sugar utilization efficiency was 75% with an ethanol yield of 86% of the theoretical maximum from the supplied mix of carbohydrates.

• An ethanol outflow concentration of more than 45 g/l was measured at a dilution rate of 0.06/hr, decreasing to 43 g/l at 0.11/hr.

[40] It remains to be seen if bacterial populations secreting в-lactamase evolve in bioethanol plants, although a wide range of antibiotics could (in principle) to be used to mitigate this resistance.

[41] Fusarium wilt is a disease that affects more than a hundred species of plants; the fungus colonizes the xylem vessels, blocking water transport to the leaves.

[42] Often (but confusingly for discussions of biofuels) abbreviated to SSF, although arguably with a claim to prior use in this area of biotechnology.

[43] Bagasse combustion in steam turbines generates electricity at 1 MWh/m3 of produced alcohol.

• Anaerobic digestion of vinasse can produce enough biogas for 0.5 MWh/m3 of produced alcohol, and both processes have been applied at full scale at distilleries.

• Laboratory studies show that anaerobic digestion would also be beneficial for bagasse, increasing the power output to 2.25 MWh/m3 of produced alcohol if the nonbiodegradable residue is burned.

• The total potential power generation from biogas and combustion routes would be equivalent of 4% of national power demand.

[44] Permanent (“constitutive”) expression of genes encoding chitinases or a ribosomal-inactivating protein confers partial protection against fungal attack.

• Enhancing lignin deposition in response to fungal or bacterial invasion is a possible multigene defense.

• Overexpressing genes encoding biosynthesis of phytoalexin antibiotics have been explored together with the introduction of novel phytoalexin pathways by interspecies gene transfer.

• Specific natural plant disease resistance genes are beginning to be identi­fied and cloned for transfer into susceptible plants.

[45] This thornless tree, known by many different names but including false koa, horse tamarind, or jumbie bean, has been included in the list of the 100 worst invaders, forming dense thickets and difficult to eradi­cate once established. Being “corralled” in energy plantations may be an appropriate use for it — but adjacent farmers and horticulturists may vehemently disagree.

[46] Corn-based manufacturing facilities at scales of 10 million up to 100 mil­lion gallons/year

[47] “It is conceivable that as the price of gasoline increases to a point greater than the price of ethanol, producers could raise the price of ethanol to equalize the prices of the two liquid fuels.”12

The operating profit was, however, entirely represented by sales of the fermenta­tion stillage delivered (by truck) to neighbors within 5 miles of the farm. The selling price of such stillage would be much depressed if a large brewery or distiller were located nearby; if no net income could be generated by these means, then the facility would run at a loss. The capture of CO2 from the fermentation was not considered because the capital cost of the equipment was too high to give a good return on the investment. A localized and small-scale production of fuel ethanol could, therefore, provide all the fuel requirements for running a farm’s gasoline-consuming opera­tions and provide a reasonable financial return as a commercial venture — but only if the “agrobusiness” was run as an early example of a biorefinery (chapter 8), pro­ducing not only ethanol but a saleable fermentation — and corn-derived coproduct.

Published in 1982, a second survey of technology and economics for farm-scale ethanol production at more than 100 gallons/hr (or, up to 1 million gallons/year) estimated a total annual cost of $1.97/gallon as a breakeven figure.14 The technical aspects of the process had been investigated in a facility at the South Dakota State University with fermentation vessels of up to 5750 l capacity. The projected price included a $0.41/gallon sales income from the wet grain coproduct, annual amortized capital cost, operating costs, and fixed costs (including insurance, maintenance, and

advances, including the introduction of corn hybrids with properties tailored for wet milling (e. g., accelerated steeping) and improved fermentor designs allowing cell entrapment were estimated to offer cost reductions of 4-70/gallon.

The “missing” analysis of lignocellulosic ethanol in the mid-1990s was (in all probability) supplied by two reviews also published in 1996.2223 Two scenarios were considered in the cost modeling: a base case and an advanced technology option (although without a precise date for implementation); the data are summarized in table 5.10. Using plausible technology for the mid-1990s, a production cost of $1.18/ gallon was computed; a fourfold increase in the capacity of the facility, together with innovative bioprocess technologies, was predicted to reduce the production costs to approximately 500/gallon.

5.1 BIOETHANOL MARKET FORCES IN 2007

5.1.1 The Impact of Oil Prices on the "Future" of Biofuels after 1980

“Economists attract ridicule and resentment in equal measures.”1

The most telling aspect of the above quote is not that it derives from a recent col­lection of essays originally published in The Economist, one of the leading opinion formers in Western liberal economic thought, but that it is the first sentence in the Introduction to that volume. Graphical representations from many economic sources share one common factor, a short time axis. In the world of practical economics, hours, days, weeks, and months dominate the art of telling the near future — for price movements in stock markets, in the profitability of major corporations and their mergers, the collapse of currencies, or surges in commodity prices. Extrapo­lations are usually linear extrapolations from a small historical database of recent trends. Pundits predict much, but whatever the outcome, their predictions may very soon be forgotten. Economic models may, with hindsight, be wildly optimistic or inaccurate, but by the time “hindsight” is raised as a debating issue, the original set of parameters may have become completely irrelevant.

The history of biofuels since the early 1970s exhibits such cycles of optimism and pessimism, of exaggerated claims or dire prognostications; a series of funding programs have blossomed but — sometimes equally rapidly — faded.2 The prime mover in that sequence of boom and bust, evangelism and hostility, newspaper head­lines and indifference, has invariably been the market price of oil. However undesir­able a driver this is in the ongoing discussions on the development of biofuels, from the viewpoint of the biotechnologist in the scientific research community, it can never be ignored.3 Even with supporting arguments based on energy security and ameliora­tion of greenhouse gas emissions, a high cost of any biofuel relative to that of gaso­line, diesel fuel, and heating oil is the main plank in the logic used by skeptics: that however worthy are the goal and vision of biofuels for the future, they simply can­not be afforded and — in a sophisticated twist of the argument — may themselves contribute to the continuing deprivation of energy-poor nations and societies while the energy-rich developed economies impose rationing of fossil fuel use and access to maintain their privileged position. Although lobbyists for the global oil industry clearly have a vested interest in continuously challenging the economic costs of bio­fuel production, an underlying fear that, whether significant climate change could be lessened by the adoption of biofuels for private transportation (a debatable but quantifiable scenario — see chapter 1, section 1.6.2) and whether “energy security” is simply a novel means of subsidizing inefficient farmers to grow larger and larger harvests of monoculture crops to maintain agricultural incomes and/or employment for a few decades more, only a clear understanding of the financial implications of biofuels can help fix the agenda for rational choices to be made about investing in new technologies across the wide spectrum of rival possible biofuel options in the twenty-first century.

It is, however, undeniable that oil price volatility can shake the confidence of any investor in bioenergy. In the two decades after 1983, the average retail price for gasoline (averaged over all available grades) was 830/gallon, but transient peaks and troughs reached 1290 and 550, respectively (figure 5.1). Slumping oil prices almost wiped out the young sugar ethanol-fueled car fleet in Brazil in the 1980s (chapter 1, section 1.2). Since 2003, the conclusion that the era of cheap oil is irreversibly over has been increasingly voiced as the demands of the burgeoning economies of India and China place unavoidable stresses on oil availability and market price.4 If accu­rate, that prediction would be the single most important aid and rationale for biofuels as a commercial reality.

The cultivation of photosynthetic microalgae under dark conditions, supplied with organic carbon, closely resembles typical microbial fermentations. Because several bac­terial species are well known as accumulators of triglycerides (oils) and esters of fatty acids with long-chain alcohols (waxes), the logical conclusion was to combine these biosynthetic abilities with that of ethanol formation to generate the precursors of tri­glycerides in microbial production systems, that is, “microdiesel” produced without any need for a chemically or enzymatically catalyzed transesterification.9 The simple bacte­rium Escherichia coli was used as host for the Zymomonas mobilis pyruvate decarbox­ylase and alcohol dehydrogenase genes for ethanol production (chapter 3, section 3.3.2) together with the gene encoding an unspecific wax ester synthase/acyl-CoA: diacylg — lycerol transferase from a bacterial strain (Acinetobacter baylyi) known to accumulate lipid as an internal cell storage reserve. The resulting recombinant could accumulate ethyl esters of fatty acids at up to 26% of the cellular dry mass in fermentations fed with glucose. Insomuch as glucose is a fully renewable carbohydrate supply (via, e. g., cel­lulose or starch), microdiesel is a genuinely sustainable source of preformed transporta­tion fuel — although the chemical engineering aspects of its extraction from bacterial cells and the economics of its production systems require further definition.

A refinement preliminary to industrial feasibility studies would be to transfer to a host capable of higher endogenous accumulation of lipids; many of these are Gram-negative species (like E. coli), and ethanol production in such species is a well-understood area of biochemistry.1011