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.