The natural structure of modern plants (83) believed to contribute to the recalcitrance of biomass to chemical or enzymatic degradation include (i) the epidermal tissue of the plant body, particularly the cuticle and epicuticular waxes, (ii) the arrangement and density of the vascular bundles, (iii) the relative amount of sclerenchymatous (thick wall) tissue, (iv) the degree of lignification (14), (v) the warty layer covering of secondary cell walls, (vi) the structural heterogeneity and complexity of cell wall constituents, such as microfibrils and matrix polymers (84), (vii) the challenges for enzymes acting on an insoluble substrate (85), and (viii) the presence of inhibitors in cell walls or generated during the conversion processes to subsequent fermentations (86). These chemical and structural features affect liquid penetration and/or enzyme accessibility and activity, and thus the overall biomass conversion costs.

Current biomass conversion technology uses chemical pretreatments to remove hemicel­luloses from the microfibrils, which in turn exposes the crystalline cellulose core rendering it more amenable to the action of cellulase enzymes. In addition, pretreatment typically breaks down the macroscopic rigidity of biomass and decreases the physical barriers to mass transport. A suite of enzymes such as cellulase, hemicellulases, and accessory en­zymes are introduced to depolymerize cellulose (saccharification). During this process, the cell walls are decomposed at the molecular level, hemicelluloses and lignins are either hy­drolyzed in place or allowed to migrate, and the crystalline cellulose structures are exposed and modified. Eventually, the polysaccharides are depolymerized to monomer sugars for fermentation. The technical barriers that contribute to the high cost of current biomass conversion processes have been identified as low sugar yields and low efficiency of en­zyme performance. To overcome these problems, improvements of these processes rely on further understanding of cell wall ultrastructure and the molecular mechanisms of enzyme hydrolysis.

The lignocellulose biorefinery is envisioned to comprise four major processing steps: 1) feedstock harvest and storage, 2) thermochemical pretreatment, 3) enzymatic hydrolysis, and 4) sugar fermentation to ethanol or bio-based products. Among these processes, chem­ical and enzymatic treatments of biomass contribute to the majority of the processing cost. An acidic chemical pretreatment step is usually conducted to depolymerize and solubilize hemicelluloses (approximately 20-40% wt/wt of biomass). This step converts hemicellu — loses to monosaccharides and oligosaccharides, which can be further hydrolyzed in the later processes. Thermochemical pretreatment of biomass has long been recognized as a critical step to produce celluloses with acceptable enzymatic digestibility. Various technologies have been developed to accomplish this goal. For example, dilute sulfuric acid pretreatment at 140-200°C renders the cellulose in cell walls more accessible to enzymes (83). For dilute acid treatments (pH ~1.5), release of mono and oligomeric sugars from hemicellulose exhibits multi-modal kinetics. It is this slow monosaccharide release phase of chemical hemicellulose hydrolysis that directly relates to the high process conversion cost (87, 88). A number of researchers (88-93) have noted that the depolymerization of hemicellulose appears to be best described as a pair of parallel first-order reactions where one takes place at a fast rate and the other at a much slower rate. Pretreatment schemes based on alkaline explosive decom­pression or organic solvent extractions have also been used with considerable success (86). The alkaline process, known as ammonia fiber expansion (AFEX), leaves the hemicellulose in place, yet renders the remaining cell walls considerably more amenable to enzymatic hydrolysis (94). At moderate pretreatment severities (95), the hemicelluloses are hydrolyzed and solubilized as monomers and oligomers; however, the yields of solubilized sugars are often unpredictable, and less than ideal (96). The improvements of chemical pretreatments now focus on increasing sugar yields and reducing the severity.

The factors that govern the pretreatment process at the level of the cell wall are not clear today. However, this process undoubtedly depends on a number of factors, such as hemicellulose composition, biomass density, the presence of non-sugar components (i. e., lignin, ash, acetyl, and uronic acids), and most importantly plant cell wall structure (i. e., types of cells, ratios of primary and secondary cell walls, as well as the macromolecular structure and arrangement of cell wall polymers).

Although it is not fully known how many enzymes are involved in cell wall deconstruc­tion in nature, over a hundred families of glycoside hydrolases (GH) have been identified in the CAZY database (http://afmb. cnrs-mrs. fr/CAZY/fam/acc_GH. html). Three general categories ofenzymes are considered necessary to hydrolyze native cell wall materials: cellu — lases, hemicellulases, and the accessory enzymes, which include hemicellulose debranching, phenolic acid esterase, and possibly lignin degrading/modifying enzymes (97). Once the hemicellulose barrier associated with cell wall microfibrils has been compromised by chem­ical pretreatments, cellulase enzymes can be used to hydrolyze the crystalline cellulose. Crystalline cellulose is hydrolyzed by the synergistic action of endo-acting enzymes known as endoglucanases, and exo-acting enzymes, known as exoglucanases. The endoglucanases locate surface sites along the glucan chain and insert a water molecule in the (3-(1,4) bond, creating a new reducing and non-reducing chain end pair. The release of cellobiose from the cellulose is thought to occur at these new chain ends and this process considered to be the rate limiting step in cellulase action, is accomplished by exoglucanases also known as the “processive” cellulases. At this time, studies of the synergistic reaction of cellulases are primarily based on assays on purified cellulose substrate such as Sigmacell, Avicel, or bacte­rial cellulose, not cell walls (98). There is no doubt that the deconstruction of the complex structures found in cell walls require a wider range of enzymes than just the cellulases; in fact, the synergistic action of many GH family enzymes as well as whole microbial cells are likely critical, and yet poorly understood (99).

Acknowledgment

This work was supported by the US Department ofEnergy, Office ofthe Biomass Program.