The combination of femtosecond lasers and the high numerical aperture optics found in mi­croscopy makes it possible to create high intensities (100 GW/cm2) with extremely modest energies (~ 100 pJ). This high intensity results in inducing a dynamic, nonlinear polarization in virtually any media located within the focal volume of the microscope objective. This nonlinear, time-varying polarization response acts as a driving force in the wave equation that can result in new source terms. These new sources can be used to create image con­trast. Because they scale nonlinearly with the excitation intensity they are strictly confined to the focal volume (no out-of-focus contributions), and in essence are naturally confo — cal. The net result — nonlinear microscopy — is a high-resolution (sub-micrometer lateral resolution, micrometer axial resolution), three-dimensional imaging modality capable of effectively probing material structure and function. While these intensities may seem ex­treme, the combination of modest energy (44) and infrared wavelengths actually results in a relatively benign excitation source. In comparison to continuous wave excitation at UV or near-UV wavelengths, delicate systems are minimally perturbed under femtosecond laser excitation.

The recently developed nonlinear microscopy has been applied to imaging biologi­cal systems, such as nonlinear signals of second (SHG) and third harmonic generation (THG) (45), coherent anti-stokes Raman spectroscopy (CARS) (46, 47). These tech­niques combine spectroscopy (chemical) and microscopy (spatial) approaches, which have particular potential in characterizing plant cell wall structures and their bioconversion processes.

Although it is envisioned that the first Phase III or lignocellulosic biorefineries will capture the low cost or niche feedstocks, eventually a significant fraction of the feedstock potential identified by Perlack and coworkers (3) will need to be captured economically. As the biorefining industry expands, process improvements will drive biorefinery capacities up.

Therefore, the longer-term feedstock supply R&D challenge is to ensure supply systems do not limit biorefinery size or consume biorefinery profits that could be used to purchase higher-cost feedstocks (see Figure 2.10). Feedstock availability, as a function of payment to the grower (4), has shown that more than 2/3 of the feedstock potential identified by Perlack and coworkers could be made available to the biorefinery at a purchase price up to about $50/ton. Adding estimated feedstock supply system costs gives a final feedstock cost of about $70/ton.

An advanced feedstock supply system will be needed to collect the large tonnages of feedstock required for large-scale biorefineries. An efficient interface between producers and the commodity biomass system is important for large-scale feedstock supply technology development. Production, harvesting, and collection systems will be widely varied, based on biomass resources and local practices. Primary research needs include storage, preprocessing, and transportation systems suited to these varied systems.

Developing value-add feedstock preprocessing and blending technologies will provide flexibility in the biomass feedstock supply system and allow suppliers to:

1 Reformat/condition different feedstocks into a common format and quality

2 Fractionate secondary co-products for local markets

3 Produce blended, large-scale commodity biomass.

Value-added preprocessing will help create a market specification for feedstocks, which will help in the transition of biomass to a large-scale commodity and ensure that feedstocks from varied sources can supply a large-scale biorefinery without process upset.

We define the collective resistance that plants and plant materials pose to deconstruction from microbes and enzymes as “biomass recalcitrance.” This trait developed in terrestrial

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

plants during evolutionary maturation, in part, as a consequence of their moving from the protection of the aquatic environment.

Although little is known about the definitive steps involved or the intermediate forms explored, modern plants possess many systems for protection. The first line of defense in most plants is the epidermis, or outer layer of the plant anatomy. In grasses, this layer usually contains dense collections of thick-walled cells, as well as specialized cells that secrete waxy or oily materials. In trees, the bark presents a considerable physical, as well as chemical, barrier to all but the most dedicated assault.

Plant defense systems extend to the structure and organization of vascular tissue and even of the cell wall. Buried in the cell wall are the elementary fibrils that harbor the cellulose core (4). Even cellulose poses a significant barrier to enzyme action, where the highly ordered and water-excluding nature of the crystallite is sufficient to significantly retard cellulase action. This point is made especially clear when considering that the processive cellulase, cellobiohydrolase II, has been estimated from kinetic data to break about 14 bonds per second (5). Cell wall microfibrils are surrounded by sheaves of hemicellulose that, in turn, is covalently linked to lignins. This matrix of heteropolymers in which cellulose is embedded is certainly the dominant reason why plant biomass has resisted low-cost chemical and enzymatic treatment schemes.

In mature tissues, there is large volume of intercellular spaces arising by splitting of adjunct cells, or by crushing of entire cells such as lacuna in vascular bundle resulting from the breakdown of protoxylem tissue. The intercellular spaces are functioned as air space or containers for various secreted materials in a living plant.

Plant cell walls and intercellular spaces are defined as the apoplast of the plant body and serve as mechanical support. Cell walls have important effects on the physiological activi­ties of plant tissues, such as absorption, transpiration, translocation, and secretion. These

functions are performed through the anatomical features of plant cell walls, particularly the pore systems. Pits, primary pit-fields, and plasmodesmata are commonly observed char­acteristics of cell walls. Pits are essentially depressions of walls. In the secondary cell wall, two adjunct cells often form opposing pits together called the pit-pair. In primary cell walls, these depressions are also called the primary pit-field, which constitutes a zone of a number of traverse “canals” penetrating the wall, called plasmodesmata. Plant cytoplasm (the sym — plast) is contiguous and connected by these plasmodesmata that allow the communication between neighboring cells. In mature tissues, secondary wall material may be deposited to form primary pits, which are small in parenchyma tissue (2-5 |xm in diameter, see Figures 3.2 and 3.3), whereas in thickened xylem tissues, deposition of thick secondary wall often forms large pits with characteristic patterns (tens of microns in diameter).

Recently, there have been significant R&D advances in dry herbaceous preprocessing that will enable the transition from the current state-of-technology bale-based system to a more cost-effective bulk feedstock system for biorefineries. However, additional advances are needed in three key areas: 1) preprocessing equipment capacity, 2) feedstock bulk density, and 3) feedstock quality. Equipment capacity and bulk density directly affect feedstock cost. Thus, they are important technical parameters to address, along with the interrelated effect on feedstock rheological properties. Furthermore, a key component of feedstock R&D is to extend preprocessing beyond size reduction to include value-added operations that improve feedstock quality for the biorefinery, such as fractionation and separation of higher-value components.

Specific research needed in this area includes:

• Developing preprocessing requirements for each feedstock type

• Understanding the relationship between biomass structure and composition for assess­ing quality-upgrade potential and developing equipment and methods to achieve these upgrades

• Understanding and controlling biomass tissue deconstruction in preprocessing and the relationships among grinder configuration, tissue fractions, tissue moisture, and grinder capacity to optimize grinder configuration for fractionation, capacity, and efficiency

• Increasing bulk densities by coupling the understanding of biomass deconstruction and rheological properties with innovative bulk compaction methods

• Understanding and controlling feedstock rheological properties resulting from prepro­cessing operations to provide a product that minimizes problems in transportation, han­dling, and queuing operations.

Advances in fluorescence labeling techniques suited to biological applications have resulted in widespread adoption of the total internal reflection fluorescence (TIRF) technique for biophysical studies (48, 49). With the recent development of photo-activated fluorescence proteins (PA-FP) (50), a new paradigm in single molecule imaging has developed. By sequen­tially imaging sparse subsets of single molecules, and localizing their centroids with molec­ular precision, composite optical images can be constructed with up to two orders of mag­nitude higher spatial resolution than with conventional methods (51, 52). This technique relies on photo-activation of the PA-FP, followed by photo-bleaching or photo-switching, such that only a sparse subset of molecular tags are excited in a given time window. If molecular fluorophores are strategically attached to relevant cellular structures, structural and chemical information of the cell and intracellular constituents may be obtained with nanometer resolution. A catalog of PA-FPs has now been developed for use in fluorescence imaging of living systems (51, 53-58).

Theoretically, any protein molecule could be expressed as a fusion protein with a fluores­cence protein (FP) then imaged by TIRF. While TIRF studies are commonplace in the life sciences and nanotechnology, this technique has only recently been adapted to the study of the structure of the plant cell wall (59). In order to specifically label the ultrastructure of the cell wall, molecular probes recognizing cell wall macromolecules have been recently reported, including monoclonal antibodies against polysaccharides (60-64) and lignin (65-67), and CBMs recognizing cell wall polysaccharides (59, 68, 69). Our previous study demonstrated, for example, that the innate binding specificity of different CBMs offers a versatile approach for mapping the chemistry and structure of surfaces that contain complex carbohydrates (59). In nature, the CBM serves as an attachment device for “harnessing” the glycoside hydrolases to their target substrates (70, 71). Several hundred putative CBMs have been identified to date and these proteins have been grouped into 43 families using amino acid sequence similarity algorithms (http://afmb. cnrs-mrs. fr/CAZY/index. html). The structures and ligand specificity of many CBMs have also been determined experimentally (71). Among these CBMs, one type, termed surface-binding CBMs, bind specifically to the planar sur­faces (1,1,0 and 1,-1,0) of crystalline cellulose Ia (72). We have used genetic engineering methods to produce labeled CBMs, for example CtCBM3-GFP is a surface-binding CBM cloned from C. thermocellum and tagged with green-fluorescence-protein (GFP). QCBM6- RFP is a polysaccharide-binding CBM cloned from C. thermocellum and tagged with red — fluorescence-protein (RFP). Figure 3.10 shows the TIRF image where green signal highlights the microfibril network structure and red signal shows the location of the cell wall matrix. In our previous study, we confirmed that CtCBM3 binds to crystalline cellulose and thus we believe that the microfibril network contains highly crystalline cellulose (59).

Systems biology research will result in improvements to feedstock and will maximize the recoverable liquid fuel per acre of land and drastically simplify the conversion process. These improvements have the potential to reduce the cost of converting lignocellulosic biomass to ethanol by about 30% for a similar sized 2000 tonnes/day facility (4). Additionally as the advanced state of technologies facilitate larger scale facilities, an additional 40% cost-of- production benefit could be realized for a 10 000 tonnes/day operation. These kinds of cost reductions are typical of conversion technologies as they mature. The oil and corn industries, among others, have seen processing costs drop dramatically over time until feedstock is the predominate cost.

It is envisioned that, through systems biology, the overall conversion process can be simplified, and capital and operating costs can be reduced (see Figure 2.11).

The advanced technology will combine several unit operations and improve the pre­treatment operation. Enzyme production and fermentation will be combined in a sin­gle organism. Thus, with enzymes produced during the saccharification and fermentation processes, the three process operations are combined into one. In addition, more robust microorganisms will eliminate the need for hydrolyzate conditioning. These technology improvements will lower the total capital cost (project cost) of a 2000 dry tonnes/day fa­cility by about 22% (44). Further capital cost reductions can be realized as these systems biology advances enable larger scale facilities that take better advantage of economies of scale.

Translational science concepts need be adapted to pursue these advancements. This ap­proach, familiar to the biomedical industry, integrates basic research (or fundamental bio­logical science) with industrial application (such as bioengineering). To meet the long-term potential of the biorefinery, significant fundamental scientific advances beyond what was described for a near-term economic competitive state of technology must be achieved (45). Additionally, it is important that these advances be implemented to realize the significant operating and capital cost reductions, so that the growth of biofuels does not stagnate due to the need to draw in higher cost feedstocks.

We know that some plants, especially non-flowering ones, evolved rapidly during the Meso­zoic Era. Ginkgos, for example, first appeared 150 million years ago and became common in the Mesozoic Era. One species, Ginkgo biloba, has been described as a “living fossil.” Certain characteristics enabled early plants to invade and become established on land. Internal ves­sels called vascular tissue circulated nutrients and water to all parts of the plant. An outer layer of waxy cuticle developed to prevent dehydration, and stomata located on the un­dersurfaces of leaves regulate respiration. Roots provide anchorage, nutrient uptake, and general interaction with the chemical/microbial systems in the soil (e. g., the rhizosphere).

New work to redirect the evolutionarily imposed protection of plants’ cell wall polysac­charides is now underway. The objective of “bioenergy plant engineering” is to use genetic tools to modify cell wall characteristics, thus permitting more-efficient chemical and en­zymatic hydrolysis processes, as well as enhanced agronomic productivity. This work will proceed phenomenologically at first — for example, mapping plant quantitative trait loci to beneficial conversion traits. However, this field will mature to a deeper understanding of the processes of cell wall synthesis and assembly, as well as enzymatic deconstruction. Eventually, these biological systems will be sufficiently understood to permit overall system engineer­ing, optimizing both cell wall production and deconstruction in ways not achievable in nature.

Cell walls from higher plants areprimarilycomposed of polysaccharides (i. e., cellulose, hemi — celluloses, and pectins), lignins, glycoproteins, and small amounts of minerals and other polymers. These polysaccharides exist in various forms as crystalline and sub-crystalline celluloses, non-crystalline hemicelluloses, and pectins. The hemicelluloses are closely asso­ciated with the surface of the rigid cellulose elementary fibril, forming a microfibril network (1). Pectins are cross-linked polysaccharides that “glue” the cell wall components together. Upon synthesis, these polymers form nanometer scale composites (i. e., microfibrils and ma­trices) as a result of temporally and spatially controlled processes which occur during plant growth and development. It is still unknown how these polymeric constituents self-assemble to form the rigid and dynamic entity embodied in the cell wall. In our current understanding

of primary cell wall structures, these associated and/or cross-linked polysaccharides form a “polymer liquid crystal” system (2).

Feedstock shrinkage (or dry matter loss) and quality reductions are major problems during feedstock storage. Shrinkage and quality reduction risks and mitigation strategies vary widely from region to region. Although rigorously developed targets for dry matter losses have yet to be developed, the general consensus is that they must be less than 5% for all feedstock types.

Specific research needed in this area includes:

• Assessing storage options and their effects on dry matter losses, compositional changes, and functional biomass changes specific to resource type and regional variables

• Establishing baselines of storage systems costs at scales from 0.8 million tons/year to 10 million tons/year to identify key cost and infrastructure issues and develop paths to minimize industrial-scale storage costs

• Understanding soluble sugar and carbohydrate loss and evaluating the feasibility of pre­venting or reclaiming those soluble sugars and carbohydrates from the feedstock during storage

• Developing cost-effective methods of large-scale bulk storage that reduce handling, elim­inate bulk flow problems, and minimize adverse physical changes that may affect plant processing.