Once we have selected a set of enzymes to be included into a cellulase system—be it a free cellulase system, designer cellulosomes, native cellulosomes, or any combination thereof—it may be desirable to consider improving the enzymes. In theory, this can be accomplished by rational design or by directed evolution. Moreover, enzyme improvement can assume different forms. We may want to increase the activity of the enzymes. Alternatively, we may want to change the optima of their physical properties, such as temperature and pH. In changing the latter properties, directed evolution can be employed with some level of efficiency, since temperature and pH can be used as a selection pressure, and the stability of the mutated enzymes can be assayed relatively easily. Indeed, some success in the literature has been reported for such endeavors (Murashima et al. 2002; Wang et al. 2005) Choi et al. 2008) . In recent studies (Hughes et al. 2006) , combinatorial and robotic handling methods have recently been instituted for improvement of cellulase activity in individual endoglucanases. However, the methodology employed a soluble chromogenic substrate, and the approach was again taken with the intention of identifying mutants with heightened activities at low pH.

Improvement in cellulase activity per se is another story. It is one thing to isolate endoglucanase mutants using soluble substrates, but it is quite another to isolate mutants of cellulases that work better on insoluble substrates. Moreover, the defining characteristic of cellulase and cellulosome action is not the improvement of a given cellulase, but how the different cellulases will work together to overcome the recalcitrant properties of the substrate. In this context, the rate-limiting step in the hydrolysis of crystalline cellulose is not the cleavage of the glycosidic bond of the cellulose chain, but the detachment of a single chain from the crystalline matrix (Bayer et al. 2007- Himmel et al. 2007, 2008a). Currently, there is really no acceptable assay for this function that can be employed for medium — or high-throughput procedures necessary for screening and selection of potent cellulases. Since there are no clear relationships between cellulase activities on soluble substrates and those on insoluble substrates, soluble substrates should not be used to screen or select for improved cellulases. Some exoglucanases, for example, show little or no activity on any substrate, but contribute substantially to the overall synergistic activity of enzyme mixtures and on insoluble cellulosic substrates. Theoretically, such assays should be based on relevant solid substrates, such as paper or plant cell walls (Zhang et al. 2006- Himmel et al. 2007). However, in practice, this has yet to be achieved in a reliable manner.

Conclusions

The structural polysaccharides in lignocellulosic biomass are a rich and renewable source of fermentable sugars for industrial production of biofuels. It is important to note that in attempting to utilize these carbohydrates at the commodity scale, we must overcome a key principle set forth in the evolutionary development of the cell wall of terrestrial plants: essential recalcitrance to deconstruction. Fortunately, progress is being made in this endeavor. Indeed, although these general process technologies are known, the key cost challenges remain the subject of considerable international research focus today. It is clear that only through dedicated, fundamental science guided by clearly defined applied objectives can such complex processes be made a reality. In this case, new and improved enzyme systems closely coupled to related process technologies, such as biomass pre­treatment, are required to provide cost-effective and large- scale quantities of liquid fuels from biomass.

Acknowledgm ents

The biomass structure, chemistry, and enzyme engineering review presented in this work was supported by the BioEnergy Science Center (a U. S. Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science); the remainder of the review was supported the Israel Science Foundation (Grant Nos. 966/09 and 159/07), by grants from the United States-Israel Binational Science Foundation (BSF), Jerusalem, Israel, and by the National Research Initiative Competitive Grant Nos. 2002-35206- 11634 and 2006-35206- 16652 from the USDA Cooperative State Research, Education, and Extension Service. E. A.B. holds The Maynard I. and Elaine Wishner Chair of Bio-Organic Chemistry.