The utility of any biomass crop as a feedstock for ethanol production will depend in large part on its chemical composition, both in terms of the amount of potentially fermentable carbohydrates and the presence of compounds that may limit the yield of these carbohydrates. Current commercial yeast strains only utilize glucose as a substrate for ethanol production. Glucose can be derived from cellulose in the cell walls of biomass species. Therefore cellulose is of greater value than hemicellulose or pectin, polysaccharides composed of numerous sugars other than glucose. However, genetically modified yeast strains and other microorganisms are under study and under development that will use a wider diversity of hexose and pentose sugars. Reduced concentrations of hemicellulose and lignin, a phenolic polymer in the cell wall, would provide benefits to an ethanol conversion system by reducing pretreatment process inputs of heat and acid prior to cellulose addition. Also, reduced lignin content of biomass should result in high concentrations of the cell wall polysaccharides, thereby increasing the potential amount of fermentable sugars. Unfortunately, composition of biomass crops is very diverse and varies due to species, genetics, maturity, and growth environment.

A survey of 190 alfalfa plant introductions in the U. S. germplasm collection found that leaves averaged 283 g crude protein (CP) kg-1 dry matter (DM) compared to only 93 g CP kg-1 DM in stem material (Jung et al., 1997). In contrast, the neutral detergent fiber (NDF) concentration of stems far exceeded that of leaves (658 and 235 g NDF kg-1 DM, respectively). These differences are reflective of the role of stems in providing an upright growth form and supporting the leaf mass. Stems of alfalfa develop extensive xylem tissue (wood) with thick

cell walls comprised of cellulose, hemicellulose, pectin, and lignin (Theander and Westerlund, 1993; Wilson, 1993). Because leaves are the site of most pho­tosynthetic activity in alfalfa, the leaves have high concentrations of enzymes and thin cell walls to facilitate light absorption and gas exchange. Representative composition of alfalfa stem material is shown in Table 5.1. Both leaves and stems have low concentrations of simple sugars and starch (Raguse and Smith, 1966), although alfalfa roots store substantial quantities of starch (150 to 350 g kg-1 DM) (Dhont et al., 2002). Lipid content of alfalfa is quite low (~20 g kg-1 DM) (Hatfield et al., 2005).

Because alfalfa is indeterminate in its growth habit, the plants increase in size and mass until harvested or a killing frost occurs. Alfalfa leaf mass increases during maturation, but at a lower rate than the increase in stem mass (Sheaffer et al., 2000). This results in a decline in leaf percentage in the total herbage harvested that can range from more than 70% leaf during early vegetative stages to less than 20% leaf when ripe seed is present (Nordkvist and Aman, 1986). During plant maturation, alfalfa leaves change very little in CP or NDF concen­tration whereas stem CP declines and NDF content increases dramatically (Sheaf­fer et al., 2000). The reason for the increase in NDF content of alfalfa stems during maturation is the addition of xylem tissue due to cambial activity (Jung and Engels, 2002). This xylem tissue has thick secondary walls and stem xylem accounts for most cell wall material when the crop is harvested.

Cell walls of alfalfa differ from grass cell wall material because of the greater pectin content of alfalfa cell walls. In very immature alfalfa stem internodes that are growing in size, pectins can account for up to 450 g kg-1 of the cell wall. Cellulose and hemicellulose contribute 340 and 120 g kg-1, respectively, to the total cell wall, with lignin accounting for the remaining wall material, in such young internodes (Jung and Engels, 2002). At this developmental stage, all of the lignin is localized in the protoxylem vessel cells and no other tissues are lignified. Once alfalfa internodes complete their growth in length, cambium meristematic activity begins to add new xylem fiber and vessel cells that lignify almost immediately. The predominant cell wall component in these tissues is cellulose (400 g kg-1 cell wall) with the rest of the cell wall material being equally divided among hemicellulose, pectin, and lignin (Jung and Engels, 2002). Phloem fiber cells also develop thickened secondary cell walls as the plant matures; however, this secondary wall is especially rich in cellulose and does not contain lignin (Engels and Jung, 1998). Lignin is deposited in a unique ring structure in the primary wall region of phloem fiber cells. With the exception of pith paren­chyma cells, all of the other tissues in alfalfa (chlorenchyma, collenchyma, epidermis, cambium, secondary phloem, and protoxylem parenchyma) do not lignify no matter how mature the stem becomes (Engels and Jung, 1998). These tissues retain only primary cell walls that are rich in pectin. The pith parenchyma will ultimately lignify, although with only marginal secondary wall development, but usually pith parenchyma cells senesce, leaving a hollow stem cavity (Jung and Engels, 2002).

The composition of the major cell wall polysaccharides and lignin also change during maturation. Hemicellulose composition shifts from slightly more than 50% xylose residues, with the remainder being primarily to mannose, in very immature elongating stem internodes to 80% xylose residues in very mature internodes (Jung and Engels, 2002). The composition of the pectin fraction shifts less dramatically, with uronic acids increasing from 60% of the pectin to 67% with decreases in galactose and arabinose content, but no change in rhamnose con­centration. The largest shift in cell wall composition due to maturity is in mono — lignol components of lignin. The syringyl-to-guaiacyl ratio increases from 0.29 to 1.01 as alfalfa stem internodes mature (Jung and Engels, 2002).

While maturity is the single most important factor that impacts composition of alfalfa, growth environment causes some additional shifts in composition. Unfortunately these environmental impacts are complex and difficult to predict. In a study by Sanderson and Wedin (1988), alfalfa herbage from a summer regrowth harvest in one year had a substantially higher NDF concentration than observed for that year’s spring harvest (538 and 476 g NDF kg-1 DM, respec­tively); however, the same plots harvested in the following year showed a small difference between summer and spring harvests (588 and 546 g NDF kg-1 DM, respectively). Acid detergent lignin (ADL) concentration of the NDF fraction was greater for summer-harvested alfalfa in both years. During the spring growth period of the second year, air temperatures were warmer and there was less rainfall than in the first year of the study (Sanderson and Wedin, 1988). Vegetatively propagated clones of individual alfalfa plants divergently selected for stem cell wall quality traits showed environmental variability when evaluated over twelve cuttings (two locations, over two years, with three harvests per year). One clone averaged 233 g kg-1 for stem Klason lignin concentration but varied in response from 198 to 261 g kg-1 over the environments tested. Another clone selected for stem cellulose concentration ranged from 396 to 467 g kg-1 for the twelve samples (Lamb and Jung, unpublished data).

In the previous study, the impacts of temperature and moisture cannot be evaluated separately. When these two environmental factors have been evaluated independently, the major effect of moisture stress alone appeared to be on amount of cell wall accumulated by alfalfa plants as opposed to changes in cell wall composition. When rainfall was eliminated using a moveable shelter and alfalfa plots were irrigated to three field capacities (65, 88, and 112% saturation), stem cell wall concentration was reduced when the alfalfa was grown under water — deficit conditions (Deetz et al., 1994). Klason lignin concentration of the cell walls was not altered due to water-deficit and concentrations of xylose, galactose, and rhamnose in the cell wall were marginally increased and glucose was decreased, under the 65% field capacity treatment. In contrast to the impact of moisture, temperature was found not to alter cell wall concentration, but did apparently influence cell wall composition. A greenhouse study where alfalfa was grown under adequate moisture conditions indicated that higher temperatures (32°C and 26°C, day and night respectively) resulted in no changes in leaf or stem NDF concentration compared to cooler growth conditions (22°C and 16°C, day and night respectively), but ADL content of the NDF was increased by the higher temperatures (Wilson et al., 1991). However, these temperature effects should be viewed with some caution because both the NDF and ADL concentra­tions observed for the greenhouse-grown alfalfa in this study were much lower than normally observed for field grown plants.