Category Archives: Biomass Recalcitrance

Chemistry and Molecular Organization of Plant Cell Walls

Philip J. Harris and Bruce A. Stone

Abbreviations used: Apif Apiofuranosyl; Araf Arabinofuranosyl; AGP, Arabinogalactan — protein; Arap, Arabinopyranosyl; pCA, p-Coumaric acid; DDFA, Dehydrodiferulic acid; DDQ, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone; FA, Ferulic acid; Fucp, Fucopyranosyl; Galp, Galactopyranosyl; Glcp, Glucopyranosyl; GlcpA, Glucopyranuronosyl; GalpA, Galac — topyranuronosyl; GAX, Glucuronoarabinoxylan; GRP, Glycine-rich protein; HCA, Hydrox — ycinnamic acid; HGA, Homogalacturonan; HRGP, Hydroxyproline-rich glycoprotein; 4-0- methyl-a-D-GlcpA, 4-0-methyl-a-D-glucopyranosyluronic acid; Rhap, Rhamnopyranosyl; RG-I, Rhamnogalacturonan I; RG-II, Rhamnogalacturonan II; Rhap, Rhamnopyranosyl; SA, Sinapic acid; XGA, Xylogalacturonan; XG, Xyloglucan; Xylp, Xylopyranosyl.

4.1 Introduction

The cell walls ofseed plants (angiosperms and gymnosperms) represent an enormous store of fermentable carbohydrate that is a potential source of ethanol. However, this carbohydrate is in the form of high molecular weight cellulose and the accompanying non-cellulosic polysaccharides, which are intimately associated both non-covalently and covalently with one another and often with non-carbohydrate polymers particularly lignins, and other polymers such as proteins, and in some situations suberin and cutin. These associations must be broken to allow access of polysaccharide degrading enzymes to their substrates during the digestion phase of bioethanol production in which fermentable sugars (monosaccharides) are released from the plant feedstocks.

Two types of cell walls are recognized, primary and secondary (1-3). Primary walls are formed while cells are still developing and enlarging and for many cell types, e. g., some parenchyma cells, the primary wall is the only wall. Primary walls are typically non-lignified and their thickness in mature cells depends on the cell type. Secondary walls, e. g., in scle — renchyma fibers, are deposited on the primary wall after the cells are fully expanded and at maturity the entire wall (primary and secondary) is typically lignified. The middle lamella, the interfacial layer between adjacent cells, which develops from the cell plate present at division, is also typically lignified. Although less common, cell types occur that have both primary and secondary walls but that are not lignified, or only slightly lignified, for example,

Biomass Recalcitrance: Deconstructing the Plant Cell Wall for Bioenergy. Edited by Michael. E. Himmel © 2008 Blackwell Publishing Ltd. ISBN: 978-1-405-16360-6

the fibers of tension wood (in hardwoods), bast (phloem) fibers from the stems of flax (Linum usitatissimum), hemp (Cannabis sativa), ramie (Boehmeria nivea) and nettle (Urtica dioica), and the hairs of cotton (Gossypium spp.) seeds (4). Many of the parenchyma cells in stems of grasses have secondary walls and the entire wall is lignified (5).

This chapter provides a background to the discussion on current methods for disasso­ciating polysaccharides from plant cell walls to make them accessible to depolymerizing enzymes (see Chapter 14) and on the specificities and action patterns of the polysaccharide depolymerizing enzymes that may be used to release monosaccharides for fermentation (see Chapter 10). In particular, we review the chemistry of cell wall polysaccharides, their associations with one another and with non-carbohydrate polymers, and models of cell wall architecture. We shall focus particularly on primary and lignified secondary cell walls of vege­tative organs of common groups of plants that have been suggested as major biomass sources for bioethanol production. Of particular importance are the herbaceous angiosperms, espe­cially the grasses, including the cereals, which form the monocotyledon family Poaceae. This family yields grain-milling residues, e. g., wheat (Triticum aestivum) bran, and crop residues, e. g., wheat straw and maize (Zea mays) stover. In addition, perennial grasses such as Miscant — hus (Miscanthus spp.), switchgrass (Panicum virgatum), giant reed (Arundo donax), and reed canarygrass (Phalaris arundinacea) have been proposed in the United States (6) or selected by the European Union (7) as being particularly promising as energy crops. The grass family forms part of a major group of monocotyledons known as commelinid monocotyledons that are characterized by the presence of ester-linked ferulic acid in their primary walls. We refer to other monocotyledons as non-commelinid monocotyledons (8).

In the rest of the angiosperms, the term eudicotyledon (or true dicotyledons) is now used to refer to most of the group previously known as the dicotyledons. Some herbaceous eudicotyledons, such as the forage legume alfalfa (Medicago sativa), have also been considered as energy crops (9). Hardwoods (woody angiosperms), e. g., poplars (Populus spp.) and willows (Salix spp.), and softwoods (coniferous gymnosperms), e. g., spruce (Picea abies), are also potential feedstocks.

Selective thermal transformation to improve thermochemical processing

Achieving the near-term economic competitiveness target with gasoline and starch-based ethanol outlined above for biomass gasification — mixed-alcohol synthesis — requires im­provements in catalytic tar and light hydrocarbon reforming to increase conversion effi­ciencies and reduce the capital costs of syngas cleanup and conditioning. However, as is the case for biochemical conversion, significant technological advances beyond this state of technology will be required to realize the ultimate potential of thermochemical conver­sion approaches for biofuels production. A strategy for accomplishing this involves moving forward with two complementary approaches:

• Pursue scientific achievements to improve yields and efficiencies and maximize process integration opportunities in existing thermochemical processes

• Implement a rigorous research program to investigate fundamental biomass thermo­chemical conversion to enable alternative processes that will help erase the lines between gasification and pyrolysis as separate technology options.

R&D efforts for breakthrough thermochemical technology should focus on the front-end processes, while the downstream unit operations continue to be optimized. Significant im­provements in catalytic gasification will be needed to increase carbon conversion efficiencies to syngas and decrease tar formation. An example of a breakthrough technology would be converting 50% of the methane produced to syngas while simultaneously increasing the throughput of the gasifier by 25%. This improved technology would reduce thermochemi­cal conversion cost by an estimated 38% over the technology listed above (46).



• Chemical fractionation • Thermochemical/catalytic/chemical

• Thermochemical pretreatment transformation with high yields and

• Catalytic modification (hetero/homogeneous) controllable selectivity


— Solid acid catalytst

— Transition metals

— Organometallics

• Biomass deconstruction

• De-polymerization

Figure 2.12 Selective thermochemical processing.

Process consolidation is needed to continue lowering capital and operating costs. The block flow diagram in Figure 2.12 illustrates the R&D required to advance thermochemical conversion technology to meet the long-term goals of the biorefinery and biofuels. The following sections describe the research needed to accomplish advanced state of technology for thermochemical conversion shown in Figure 2.12.

Fermenting cell wall sugars: the stage is set for systems/synthetic biology

It is absolutely critical that the entire suite of sugars produced from all types of biomass be effectively converted to ethanol (or other products) by the fermentative microorganism — the ethanologen. A particular concern is the conversion of five-carbon sugars, primarily xylose and arabinose from grasses and hard woods. Desired characteristics for the ideal ethanologen include the following: it ferments all biomass sugars equally well (glucose, xylose, arabinose, galactose, mannose, and even sucrose); it resists toxic compounds produced during pre­treatment (furfural, hydroxymethyl furfural, acetic acid, and soluble phenolics); it ferments high concentrations of sugars likely to be produced from high-solids pretreatments; and it produces fermentation beers with byproducts credits intact (9).

Realizing the potential of cellulosic biofuels may be facilitated by applying a new gener­ation of genomic research tools. Metabolic engineering is now used routinely to develop microbial biocatalysts. Key approaches are the targeted manipulation of their metabolic pathways, or the introduction of new ones, with the goal of improving cellular properties or directing the synthesis of metabolic products with commercial value.

There are many examples now where metabolic engineering has improved the conver­sion yield, productivity, product concentration, and economic feasibility of an industrial bioprocess (10). One particularly relevant example is the success in extending the sub­strate utilization range of yeast and bacteria to include the pentose sugars derived from the hemicellulose fraction of biomass for conversion to fuel ethanol. Other examples include the introduction of genes that permit microorganisms to metabolize cellulose, starch, xylan, lactose, cellobiose, and sucrose. Other work has improved microbial growth rates and yields, nutrient uptake, and strain stability, or has reduced the overflow metabolism that causes the accumulation of inhibitory organic acid byproducts.

These efforts have provided a greater understanding of microbial physiology and the com­plexity of the interactions between metabolic pathways and their regulatory networks. A key discovery to emerge from these studies is that flux control is often distributed over several reactions in a pathway, rather than at a single “rate-limiting” step. Consequently, simulta­neous and coordinated overexpression of all the genes encoding a metabolic pathway may be necessary to increase metabolic flux without the detrimental accumulation of metabolic intermediates. Sophisticated in-silico models of complex metabolic networks are now used to define the minimal set of genes needed to optimize growth or product formation under particular conditions (11).

The “genomics revolution” has opened a whole new dimension to metabolic engineering. More than 800 microbial genomes have been sequenced thus far, representing enormous metabolic potential as a source of novel genes for strain development. Not too surprisingly, many enzymes catalyzing the same reaction in different microorganisms show widely vary­ing kinetic properties. Furthermore, in vitro enzyme kinetics may not predict the in vivo activities of a complex pathway, making rational selection of best-pathway genes difficult. Combinatorial assembly of divergent homologs, coupled with strain selection and evolu­tionary adaptation, can overcome many of the limitations with rational gene selection.

The emerging field of synthetic biology now makes it possible to synthesize and assemble DNA fragments into modular cassettes that encode an entire metabolic pathway, synthetic chromosomes, and even whole genomes (12). The transplantation of a whole genome from one species of bacteria into another has recently been demonstrated and represents a major step toward developing customized microbial biofactories (13).

Microbial strain development historically relied almost exclusively on mutagenesis and selection to identify strains with superior traits, and the success of this approach is still evident today in the commercial production of amino acids, antibiotics, solvents, and vita­mins. However, a systematic integration of the data generated by genomics, gene expression profiles, proteomics, and metabolomics offers the promise that we may develop a cohe­sive understanding of cellular metabolism sufficient to guide rational strain design. The new methods of synthetic biology now provide us with the means to introduce vast ge­netic diversity into a microbial host. And when combined with selection, high-throughput screening, and evolutionary adaptation, synthetic biology will allow us to identify those combinations of genes that optimize bioprocesses.

The macrofibril and elementary fibril

Using AFM, we recently observed composite structures appearing on the inner surface of parenchyma cell walls. These structures appeared to be bundles of fibrils, termed the macrofibril (9). The size of the macrofibril varies from 50 to 250 nm in diameter. Figure 3.8


Figure 3.8 Atomic force micrograph of the surface structure of maize parenchyma shows the macrofibril branching at the end.


Figure 3.9 High-resolution atomic force micrograph of maize parenchyma wall shows the detailed surface structure of microfibrils. The diameter of individual microfibril is only about 3-5 nm.

shows a large macrofibril “branching” or untangling at the end to form a set of smaller parallel fibers which appears to be the microfibrils. The macrofibril is also observed in our recent study on fresh parenchyma cells. The morphology of fibers in the macrofibril appears to be faceted, which differs from that of the microfibril (Ding, unpublished data).

The elementary fibril is the native form of the plant cell wall cellulose. Direct characteri­zation of the molecular structure of the elementary fibril has not been reported. Figure 3.4 is the recent model we proposed (9). In this model, the 36 cellulose chains are arranged in an Ip-like structure, forming a hexagonal geometry in cross section with approximate di­mensions of 3 x 5.5 nm. For this model, there are three distinct layers of chains: 6 group-C1 core chains, 12 group-C2 transition chains, and 18 group-C3 surface chains. The elementary fibrils are considered to be highly crystalline. However, due to the fact that half of the cel­lulose chains are surface chains and their interactions with the cell wall matrix polymers, elementary fibrils are expected to have some structural disorders.


Over the years, significant progress has been made in reducing cellulose enzyme costs, and very impressive cost reductions have been reported recently (37, 38). Although this progress is a major step toward the economic competitiveness of the overall biochemical process, further cost reductions in cellulose enzymes are still needed. Essentially there are two ways to reduce cellulose enzyme costs, which are typically expressed on a normalized cost of gallon-of-ethanol-produced basis: increase enzyme-specific activity or decrease enzyme production costs. Although both enzyme cost reduction approaches are useful and necessary to accomplish the cellulase enzyme cost reduction goal, increasing the enzyme-specific activity has the added benefit of reducing saccharification time, which also increases the effective utilization of capital. Specific research needed to accomplish these objectives is as follows:

• Understand cellulase interactions at the plant cell wall ultrastructural level to optimize hydrolysis processes, enzyme kinetics, and, ultimately, cellulase use and cost

• Determine how cellulase enzymes move along the cellulose chain and the roles of enzyme substructures

• Conduct targeted substitutions of enzyme components to increase specific activity guided by molecular modeling of cellulase/substrate interactions

• Identify enzyme production processes and logistics to minimize processing and trans­portation costs of enzyme products.

Chemistry of cell wall polymers

4.2.1 Chemistry of cell wall polysaccharides

Cell wall polysaccharides are of two types: the first is the stereo-regular homopolymer type among which cellulose that makes up the microfibrillar phase of all cell walls is the pre­mier example, and the second comprises the stereo-irregular, non-cellulosic polysaccharides represented by the (1—>3,1—>4)-(3 — D-glucans, heteroxylans, heteroglucans, heteromannans, and the pectins (pectic polysaccharides) which are found in the matrix phase of the wall. The chemistry and occurrence of these two polysaccharide types is described in the fol­lowing sections. Table 4.1 summarizes the cell wall components of the vegetative parts of target “cellulosic” ethanol feedstocks. It should be noted that the stereo-irregular polysac­charides other than the pectins are loosely referred to as hemicelluloses in many publications. We prefer to use the term non-cellulosic polysaccharides to describe collectively all these molecules and to refer to the individual polysaccharides specifically by their descriptive chemical names (8). The extension of the term to “hemicellulase” further obfuscates the

Table 4.1 Compositions of the cell walls of vegetative parts of "cellulosic" ethanol feedstocks

Wall type

Plant group

Primary walls

Secondary walls



e. g., Hardwoods [e. g., willows (Salix spp.) Eucalyptus spp.] and


Fibrillar phase: cellulose Matrix phase: Pectic polysaccharides > xyloglucans > heteroxylans and (galacto)glucomannans (minor)


Fibrillar phase: cellulose Matrix phase: 4-O-methyl — glucurono-xylans > glucomannans

Ester-linked ferulic and small proportions of p-coumaric acid in "core" Caryophyllales. Known to be linked to RG-I side chains in Amaranthaceae

Lignins: Syringyl and guaiacyl; small proportions of p-hydroxyphenyl units. Acylated with p-hydroxybenzoic acid in Salicaceae and acetic acid in Hibiscus cannabinus


Non-commelinids e. g., Sisal (Agave sisalana)

Fibrillar phase: cellulose Matrix phase: As eudicotyledons

Fibrillar phase: cellulose Matrix phase: As eudicotyledons


e. g., Grasses and cereals

(Poaceae), palms (Arecales)

Fibrillar phase: cellulose aMatrix phase:

Glucuronoarabinoxylans (GAXs) > pectic polysaccharides and xyloglucans

[(1^3,1^4)-B-glucans in some Poales families, including


Fibrillar phase: cellulose Matrix phase:

Glucuronoarabinoxylans > (galacto-)glucomannans (minor)

Ferulic, small proportions of p-coumaric, and sometimes sinapic acids, ester-linked to GAXs

Lignins: As in eudicotyledons. Acylated with p-coumaric acid in Poaceae and Musaceae, and with p-hydroxybenzoic acid in Arecales


e. g., Coniferous


gymnosperms, e. g., spruce (Picea abies)

Fibrillar phase: cellulose bMatrix phase: As eudicotyledons

Ester-linked ferulic and p-coumaric acids in all families

Fibrillar phase: cellulose Matrix phase:

(Galacto-)glucomannans > 4-O — methyl-glucuronoarabinoxylans Lignins: Usually mostly guaiacyl units;also p-hydroxyphenyl units in compression wood

a The primary walls of palms (Arecales) are an exception; their matrix polysaccharides are pectic polysaccharides > xyloglucans > glucuronoarabinoxylans (minor).

b Detailed dataon the matrix polysaccharides are available only forconifers (Coniferales) and Ginkgobiloba (Ginkgoales).

situation since the enzymes hydrolyzing non-cellulosic polysaccharides are specific and cannot be grouped together (see Chapter 10).

Catalytic gasification and pyrolysis

Since the beginning of coal gasification, catalysts have been sought that would improve carbon conversion to products and increase gasification rates, while minimizing temperature to increase process efficiency. Alkali metals have long demonstrated catalytic activity in steam gasification of solid fuels, and metal-based catalysts — particularly nickel-based materials — are active and effective for hydrocarbon reforming. More R&D along these lines is needed to achieve higher carbon conversions and increased efficiencies in gasification. In the area of pyrolysis, catalytic pyrolysis processes are needed to improve selectivities to more desirable compounds. Lignin utilization

Integrating and using lignin residues produced from biochemical-based biorefineries will be key factors in establishing the long-term viability of lignocellulosic biorefineries and maximizing biomass use for fuel production. First-generation biorefineries, call for the lignin to be simply burned to supply the heat and power needs of the biorefinery. Although this is economically viable in the near-term, ultimately because lignin is a complex but lower-value biomass component, it is essential that new technologies increase its value to enhance the competitiveness of integrated biorefineries. Selective thermal transformation of fractionated biomass

A range of alternative conversion options is envisaged through the fractionation of biomass into specific components. A narrower, more uniform biomass fraction opens the possibility of developing thermochemical conversion options with high yields and selectivities.

The Biorefinery

Thomas D. Foust, Kelly N. Ibsen, David C. Dayton, J. Richard Hess, and Kevin E. Kenney

2.1 Introduction

A significant challenge for the future will be meeting the world’s mobility and chemical needs as populations and mobility needs grow. Currently, crude oil supplies almost all of the world’s transportations’ fuel need and a significant portion of the material and chemical needs (1). Overdependence on crude oil is leading to concerns about national energy security and short — and long-term price stability for both transportation fuels and commodity chemicals made from crude oil. Additionally, the everincreasing worldwide emissions of CO2 from the transportation sector and the effect this is having on global climate change further increase concerns of over-reliance on crude oil.

Biomass, as the only source of renewable carbon, shows great promise for the large-scale economical production of renewable transportation fuels and chemicals. Biomass is an extremely abundant resource that can be produced in agriculture, forestry, and microbial systems. Biomass can also be captured from waste sources such as urban wood residues. Worldwide production of terrestrial biomass has been estimated to be on the order of 200 x 1012 kg (220 billion tonnes) annually. To put it in perspective, the total energy content from this amount of biomass (via heat of combustion analysis) is approximately five times the energy content of the total worldwide crude oil consumption (2).

A recent study (3) that looked specifically at US production capability showed the po­tential for the sustainable production of 1.3 billion dry tons per year of biomass from forest and agricultural lands without negatively impacting food, feed, and fiber production while still meeting export demands. Figure 2.1 shows the portion of this potential that could be produced from forest and agricultural lands by the middle of the next century with ag­gressive policies and economic incentives to maximize biomass production. The underlying assumptions in Figure 2.1 are that both agricultural resources and perennial energy crops are produced from agricultural land including some currently protected, or reserved, land with the forest resources being produced on forest land. Additionally, the agricultural resource potential includes grains that would be available for biofuels production.

Table 2.1 shows the transportation fuel production potential from the amount depicted in Figure 2.1. Ethanol was chosen as the representative biofuel because very accurate yield data exists for the various categories of feedstocks. To develop the yield numbers listed in Table 2.1, traditional corn dry-mill ethanol technology yields were used for the grain

Biomass Recalcitrance: Deconstructing the Plant Cell Wall for Bioenergy. Edited by Michael. E. Himmel © 2008 Blackwell Publishing Ltd. ISBN: 978-1-405-16360-6


Figure 2.1 Agricultural and forest land resource potential.

resource potential, biochemical conversion process yields were used for the agronomic resource potential, and thermochemical process yields were used for the forestry resources. Biochemical — and thermochemical-to-ethanol yields are from Foust and coworkers (4).

In 2006, motor gasoline demand in the United States was approximately 143 billion gallons per year (5), so the ultimate potential for biofuels would be to supply approximately 60% of current gasoline demand on an energy basis. Although detailed data on worldwide biofuels potential does not exist, it is reasonable to assume the potential on a worldwide basis would be similar to the United States. However, only looking at biofuels as a replacement for existing transportation fuel usage is somewhat shortsighted and misleading. If the goal is to reduce dependence on crude oil, it is important to look at biofuels as a part of the overall solution to reducing worldwide dependence on imported oil. Scenarios have been developed in which sustainable development of biofuels, in conjunction with vehicle efficiency gains and smart growth, have been shown to be capable of virtually eliminating gasoline demand by the year 2050 (6). Given this significant potential, the challenge is to quickly deploy biofuels technology in an economically viable manner at a large scale.

Table 2.1 Total ethanol production potential


Tons available (million dry tons/yr)

Yield per ton of biomass (gal./ton biomass)

Total EtOH potential (billion gal./yr)





Agriculture residues


90 gal./ton


Perennial energy crops


90 gal./ton


Forest resources


94 gal./tonb


Total — (volume basis)



Total — (energy basis) — gallon


gasoline equivalence (GGE)

a Total grains available for biofuels production in high case from Perlack etal. (3) minus soybeans. b Total mixed alcohol production (80 gal./ton being EtOH).

The biorefinery concept is commonly presented as the method for large-scale deployment of biofuels. The term biorefinery was established in the 1990s (7) and has been refined many times over the years. The National Renewable Energy Laboratory’s (NREL) web site defines biorefineries (8) as follows:

A biorefinery is a facility that integrates biomass conversion processes and equipment to pro­duce fuels, power, and chemicals from biomass. The biorefinery concept is analogous to today’s petroleum refineries, which produce multiple fuels and products from petroleum. Industrial biorefineries have been identified as the most promising route to the creation of a new domestic bio-based industry. By producing multiple products, a biorefinery can take advantage of the differences in biomass components and intermediates and maximize the value derived from the biomass feedstock while also being able to adapt to changing market conditions. The high-value products enhance profitability while the high-volume fuel helps meet national energy needs.

Sometimes in the literature the term “integrated biorefinery” is used (9) and is generally used in the context that a number of unit operations or technologies are used in an integrated manner to convert biomass to fuels and chemicals, so essentially the terms “biorefinery” and “integrated biorefinery” are interchangeable in the literature.

A good explanation of the stages or phases of biorefineries is provided by Kamm and Kamm (10) and Van Dyne and coworkers (11). They explain the progression of biorefiner­ies in three phases as the technology develops to move from the simple, easily processed feedstocks at lower volumes to the more difficult to process lignocellulosic biomass feed­stocks at higher volumes. An example of a Phase I biorefinery would be an existing corn dry-mill ethanol plant. It uses corn grain as the feedstock and a fairly straightforward set of conversion technologies that limit capital costs but put fairly confined constraints on production flexibilities and co-product production capabilities. In fact, most corn dry-mill ethanol plants are limited to an animal feed co-product, distiller’s dry grain (DDG), as their only co-product in addition to ethanol. A dry mill has very limited capabilities to change its product mix to adapt to changing market conditions. However, a major advantage of corn dry-mill technology is its low capital cost. A cane sugar-based ethanol plant produc­ing ethanol and food sugar, as currently exists in Brazil, is another example of a Phase I biorefinery.

An example of a Phase II biorefinery is an existing corn wet-mill plant. Although a wet — mill plant processes corn grain, it has more operational flexibility compared to a dry mill to produce a multitude of products such as ethanol, starch, high fructose corn syrup, corn oil, and corn gluten meal. The product mix in a corn wet mill can be varied to provide the highest economic return based on current market conditions. However, this co-product and product flexibility commands higher capital costs so wet mills tend to be twice as large, on average, compared to current dry mills to take advantage of economies of scale. Yet, even with the larger sizes, wet mills tend to have about a 10% higher capital cost per bushel of corn processed than dry mills (12). The Phase I and II biorefinery discussion does not need to be limited to corn wet and dry mills, an existing pulp and paper mill could be considered another example on a Phase I biorefinery that produces primarily a single project. A Phase III biorefinery would be essentially an integrated biorefinery capable of producing fuel(s) and other products from various feedstocks which would include lignocellulosic feedstocks.

Ethanol production has seen tremendous growth since 2000 primarily from new biore­finery construction and, to a lesser extent, from expanding the capacity of the existing
biorefinery industry (13). Essentially, all of the new ethanol production capacity being added is Phase I biorefineries, corn dry mills, suggesting investors favor lower capital costs over product flexibility. As an important historical reference, this indicates that lower capital costs should be favored over product flexibility for developing Phase III biorefineries.

Although corn grain and other starch — or sugar-based feedstocks processed in Phase I or II biorefineries can start the transition from sole dependence on petroleum for transportation fuels, as shown in Table 2.1, grain- and sugar-based biofuels have limited potential. Some studies (14) have listed the ultimate potential of biofuels considerably above the 9.2 billion gallons shown in Table 2.1, but the general consensus is that for biofuels to have significant production potential at the current scales of petroleum-based transportation fuels, the technology must be advanced to Phase III biorefineries that economically convert abundant lignocellulosic feedstocks into fuels and chemicals. The next sections describe the challenges and technology needed to achieve economic viability of Phase III biorefineries.

The microfibril

It is thought that the microfibril contains a single elementary fibril surrounded by hemicel — luloses (see Figure 3.9). The dimensions of microfibrils determined by electron microscopy, and recently by AFM, range from 2 to 3 nm to 20 to 50 nm (13). This apparent size vari­ation of the microfibrils may be due to the different plant species and tissue types, but is more likely due to the sampling errors and inaccurate measurements made using imag­ing techniques with limited resolution. Current biochemical evidence has shown similar biosynthesis mechanisms (e. g., highly gene homology) for all higher plants (14). There is no evidence to suggest that the elementary fibril is synthesized differently among higher plant species. In contrast, the microfibrils are dynamic structures that could be modified post-formation depending on their interaction with the matrix polymers. The diameter of the microfibrils could be different for different tissues, different cell wall types, or even for different cell wall lamellae.


Developing a robust, commercially viable biocatalyst (or microorganism) capable of fer­menting a large percentage of both the hemicellulose sugars as well as glucose to commer­cially viable ethanol titers at commercially viable fermentation times is a significant goal. Foust and coworkers (4) define near-term commercially viable targets as capable of fer­menting 85% of hemicellulose sugars and 95% of glucose to a concentration of at least 6% ethanol in 3 days in combined hybrid saccharification and fermentation, while maintain­ing the solids concentration necessary for the ethanol concentration target above. Specific research needed to accomplish these objectives is as follows:

• Identify strain candidates that exhibit superior “wild-type” performance

• Use metabolomics, proteomics, and other tools to understand metabolicbottlenecks in the carbon assimilation pathways that limit pentose sugar uptake and the ability to withstand fermentation inhibitors such as organic acids, low pH, and increased temperature

• Extend “omics” studies to identify and understand secondary pathway limitations related to reaction cofactors and regulation of metabolism

• Increase pentose uptake rates by applying protein and metabolic engineering to increase sugar transporter efficiency, pentose specificity, and expression

• Improve strain robustness by manipulating cell membrane composition to reduce its permeability to organic acids and improve its temperature stability

• Use a combination of metabolic engineering, mutagenesis, and long-term culture adap­tation strains on actual pretreatment hydrolyzate to achieve targeted fermentation per­formance

World Oil Price (2006 dollars/barrel)


• Perform parametric analysis of such factors as lignin redeposition and the detrimental effects this can have on enzyme kinetics to minimize these effects

• Use information about the enzyme capabilities and fermenting strain’s performance to develop and test strategies for efficiently integrating enzymatic hydrolysis with biomass sugar fermentation to maximize cellulose hydrolysis and sugar fermentation rates and yields

• Quantify the effects of enzyme loading, strain inoculation time, and inoculum charge on batch process performance

• Use reactor designs and operational schemes to maximize the solids loading and conver­sion of cellulose and other carbohydrates to ethanol. INTEGRATION/PROCESS ENGINEERING R&D NEEDS Finally, it is important to consider all the individual unit operations in the context of an integrated process. Although there is general agreement in the literature about necessary per­formance targets for individual unit operations as outlined above, overall integrated process targets are more difficult to define. Essentially, the overall process must be economically vi­able in the fuel marketplace, and the process must be demonstrated at some reasonable pilot scale of continuous reliable operation. Economic viability in the fuel marketplace is largely a function of gasoline prices, which are driven by crude oil price projections. Figure 2.6 shows the latest DOE Energy Information Agency 2008 Annual Energy Outlook projections for world crude oil prices out to 2050.

Foust and coworkers (4) picked $1.07 in 2002 dollars as a production cost target, which includes an IRR (internal rate of return) of 10% as an economically viable target for cellulosic ethanol. The rational provided for this target was based on historical fuel ethanol prices as shown in Figure 2.7. The $1.07 in 2002 dollars ($1.31 in 2007 dollars) per gallon value

Подпись: 0 per gallon 350 300 50

Historic fuel ethanol prices


1985 1990 1995 2000 2005 2010

Figure 2.7 US list prices for ethanol.

represents the low side of the historical fuel ethanol prices, and, given historical price data, cellulosic ethanol would be commercially viable at this production cost.

In addition, the target price of $1.31 per gallon of ethanol is also in line with current gasoline rack, or pre-tax, prices. To compare the target ethanol price with the price of gasoline on an “apples to apples” basis at the pump, the ethanol price must be adjusted as follows:

1 Adjust the ethanol price from dollars per gallon of ethanol to dollars per gallon of gasoline equivalent by correcting for the two-thirds lower energy content of ethanol compared to gasoline. This increases the $1.31 per gallon ethanol to $1.96 per gallon gasoline equivalent.

2 Adjust ethanol price from plant gate to retail price. The price of gasoline includes, on average, $0.40 per gallon for taxes and $0.23 for distribution. Assuming the same costs for ethanol gives it a retail price slightly higher than that of gasoline when oil is at $65 per barrel, as shown in Figure 2.8. This price at the pump analysis does not assume any subsidy for ethanol.