Hemicellulose is a heterogeneous polymer of different polysaccharides mainly present in the secondary cell wall. The most common type of polysaccharide is xylan which co-exists with other polysaccharides such as arabinan, galactan and mannan. Arabinan is the second most abundant polysaccharide present in hemicellulose after xylan in most plants. Other polysaccharides are often present in small amounts. The proportions of these polysaccharides differ significantly depending on their source and method of extraction (Chandrakant and Bisaria 1998).

The crystallinity index of hemicellulose is lower than cellulose, primary because of the highly branched structure and presence of acetyl group within the polymer chain. In addition, the degree of polymerization of hemicellulose is between 150-200 monomeric units (Harmsen et al. 2010).

Carbon Sequestration and Soil Organic Carbon Storage

Grogan and Mathews (2001) reviewed available soil C sequestration models and concluded then that the CENTURY model seemed to have the best potential for adaptation to bioenergy crop systems because of its integrated plant-soil approach and the availability of specific forestry subroutines. Not surprisingly, the model has been incorporated into many crop growth models, such as EPIC and DAYCENT to simulate SOC dynamics along with crop growth (Del Grosso et al. 2005; Izaurralde et al. 2006).

Available lands for biofuel production are very limited; therefore, meeting the feedstock needs of the bioenergy industry may require the conversion of agricultural lands to bioenergy crops. Land-use change consequently affects the ecosystem C balance. Corn, soybeans, and wheat are the three major crops with the highest production among food crops in U. S., and switchgrass and Miscanthus are two second-generation biofuel crops with the potential to produce a large amount of biofuel feedstocks and mitigate carbon emissions (Parrish and Fike 2005; Tilman et al. 2009; Pimentel et al. 2010). Davis et al. (2011) used the DAYCENT model to estimate the amount of N leaching and storage of soil C when replacing lands currently growing corn with perennial biofuel grasses. Overall, the model predicted a significant decrease in N leaching up to 24% and an increase in SOC up to 2.8% when land use changed from corn to switchgrass. These results are consistent with field studies showing that perennial grasses like switchgrass can store 1.1 Mg C ha1 yr-1 in the upper 1 m of the soil on conservation reserve program lands (Gebhart et al. 1994). In another field study, McLaughlin and Walsh (1998) reported that soils under switchgrass production had SOC sequestration rates reaching 20 to 30 times greater than soils under annual row crops. Furthermore, McLaughlin et al. (2002) revealed that switchgrass grown on bioenergy research plots could add 1.7 Mg C ha-1 yr-1.

Biomass generated syngas (or producer gas) contains many impurities, which must be removed through conditioning to a level acceptable to downstream applications for fuels, chemicals and power production. H2/CO ratio and compositions in the biomass-generated syngas are also generally lower than required for the syngas conversion into fuels and chemicals. The improvement in gas composition and reduction in impurities are accomplished through conditioning of syngas.

Higher substrate concentrations affect the agitation, lower oxygen transfer rates and reduce the availability of enzymes, hence limiting the rate of cellulolytic enzyme synthesis (Oashima et al. 1990). The enzyme synthesis rate is directly proportional to substrate intake. Cellulose and xylans are insoluble polymers that could lead to a longer lag phase during enzyme fermentation. A supplement of soluble sugars alongside lignocellulosic biomass plays an important role in inducing enzyme production. Both the carbon and nitrogen sources are equally important requirements in the enzyme synthesis. Urea and aqueous ammonium hydroxide are potential nitrogen sources.

An economically efficient biorefinery could be expected to require a steady flow of feedstock throughout the year. The logistics of providing a daily flow of several thousand Mg of bulky biomass could be challenging. Hwang (2007) enhanced a model (Tembo et al. 2003; Mapemba et al. 2007; Epplin et al. 2007; Mapemba et al. 2008) designed to approximate a just-in-time system for delivery of feedstock.

Providing a continuous flow of feedstock could be a major challenge for a biofuel production system that relies exclusively on switchgrass for feedstock. Most published estimates of switchgrass production costs assume that switchgrass would be harvested once per year when yield per hectare is maximized resulting in a narrow harvest window. A wide harvest window could reduce the fixed costs of harvest machinery per Mg of feedstock relative to a narrow harvest window and reduce storage cost. However, switchgrass harvestable dry matter yield and fertilizer requirements differ across harvest month (Haque et al. 2009; Haque 2010). The maximum expected dry matter yield of switchgrass grown in the U. S. Southern Plains is obtained by harvesting in either September or October (Table 10). In most years, harvest during April, May, and June would damage the plants and result in lower expected yields in subsequent years. The downside of an extended harvest window is that the expected yield from harvest in July is approximately 80 percent of maximum, and if switchgrass is left to stand in the field, dry matter losses of 5 percent per month are expected from November through March. Established stands of switchgrass that are harvested in July are expected to require about 90 kg/ha/yr of nitrogen to achieve the plateau yield, whereas fields harvested from October through March are expected to require approximately 67 kg/ha/yr (Haque 2010).

Conventional budgeting is necessary but insufficient to determine if the cost savings from extending harvest over nine months are sufficient to offset the losses in harvestable yield. A more comprehensive modeling approach is required to fully evaluate these tradeoffs. To address these tradeoffs, models may be constructed that encompass the entire chain of economic activities from acquisition of land use to delivery of baled switchgrass. Modeling may be conducted to determine if the additional fertilizer cost and the lower yield from an extended harvest window can be offset by the reduction in cost resulting from fewer harvest machines. The model optimally selects the number of harvest machines and the quantity of biomass to harvest by month and county during the potential July through March harvest window.

Bio-oil contains 30-40% oxygen (shown in the Table 5), which is similar to oxygen content in the original biomass that bio-oil is derived from. In comparison, petroleum crude oil and heavy fuel oil contain less than 1% oxygen. The oxygen in bio-oil is distributed in the form of many functional groups. High oxygen contents results in many of its undesirable properties such as low energy content and high acidity. Removal of oxygen from the bio-oil is one of the biggest barriers of using bio-oil especially for the production of drop-in hydrocarbon fuels.

High Acidity

The pH of bio-oil is 2.5 (shown in the Table 5), which makes it very acidic and highly corrosive. The acidity of bio-oil is due to presence of carboxylic acids such as acidic acid and formic acid (Zhang et al. 2007).

Switchgrass holds great promise as a valuable fuel crop for cellulosic ethanol production with pretreatments discussed earlier such as dilute sulfuric acid, sodium hydroxide, soaking in aqueous ammonia, ammonia fiber explosion, hot water, and lime pretreatment, etc. (Yang et al. 2009; Digman et al. 2010; Xu et al. 2010; Tao et al. 2011). Based on the discussed requirements for cellulosic ethanol process integration, soaking in aqueous ammonia (SAA) pretreatment may be the most feasible for lignocellulosic feedstock such as switchgrass (Isci et al. 2008; Isci et al. 2009). However, for the SAA pretreatment, a pressure vessel is required. The design of the pressure vessel is dependent on the concentration of aqueous ammonia, operating temperature, switchgrass loading, and a ratio of switchgrass to aqueous ammonia. The vapor pressure exerted by 15% (w/w) aqueous ammonium hydroxide at 80°C is approximately 31.5 psi (absolute). After the pretreatment, ammonium hydroxide may be recovered through condensation followed by lignin separation in the pretreated solvent using a filter press. The recovered lignin may then be used for power generation or have industrial importance in making biomaterials and paints (Gargulak and Lebo 1999; Lora and Glasser 2002; Keshwani and Cheng 2009; Laser et al. 2009). After recovering the lignin, the filtered water should be recycled for use in both washing SAA-treated solids after the pretreatment or to make up the ammonium hydroxide concentration after the condensation of recovered ammonium hydroxide. Recovered ammonium hydroxide concentration could be maintained up to 35% (w/v) in the separate vessel.

After SAA pretreatment, further processes could be approached in two different ways using either SHCF or SSCF for cellulosic ethanol production. As mentioned earlier, SSCF has an advantage of requiring a minimal number of vessels compared to SHCF. However, the consideration of downstream processing and energy requirements during the process would help in economical process integration for the cellulosic ethanol production. The addition of reverse osmosis between the hydrolysis step and the fermentation step would be beneficial in the concentration of the hydrolyzed sugar slurry thus minimizing the energy requirement for both ethanol fermentation and distillation. Moreover, the reverse osmosis is often more energy efficient when compared to conventional evaporation techniques (Madaeni et al. 2004). Gul and sek (2009) have mentioned that the concentration of 15% (w/v) sugar syrup to 65% (w/v) requires 86% less energy using reverse osmosis and evaporation technique compared to evaporation technique alone. The enzyme hydrolysis yields up to 10% (w/v) sugars syrup using SAA-treated lignocellulosic biomass. The addition of a reverse osmosis step following enzyme hydrolysis would allow increasing the concentration of hydrolyzed lignocellulosic sugar syrup from 10% (w/v) to 20% (w/v). The concentrated sugar syrup would influence both the fermentation and distillation steps in terms of energy saving and increasing ethanol productivity.

Figure 6 shows the overall process scheme for ethanol fermentation of glucose and xylose illustrated in separate fermenters using S. cerevisiae and P. stipitis, respectively. The dissolved chemicals in the spent broth after the ethanol distillation could be recovered using evaporation. The evaporated water vapor could be condensed and recycled to the enzyme hydrolysis step.

The future widespread planting of switchgrass as a bioenergy crop is a highly multidimensional and complex issue. Apart from the highly important social and economic components, which this chapter does little more than make mention of their existence, there is room for further work on a number of aspects that may strengthen the argument for its incorporation into the landscape and the fabric of near-future bioenergy portfolios. Critical pressing and future research can easily be tied to target areas discussed in this chapter. More specifically, improvements in (a) biomass and ethanol outputs, (b) other growth aspects and plant protection, and (c) multi-use possibilities, will simultaneously require recognition of environmentally sustainable outcomes (Box 1). The breeding and biotechnological programs essential to these endeavors (see Aguirre et al. 2012) will benefit from concurrent genomics efforts (e. g., Palmer et al. 2012; Wang et al. 2012).

Switchgrass is considered a prime candidate as a second generation biofuel feedstock because it can produce more ethanol per unit area and triple the net energy content than ethanol derived from corn grains (Bouton 2007). However, current estimates show that it requires 45 percent more fossil fuel energy to yield one liter of ethanol from two and a half kg of switchgrass feedstock than the energy in that one liter of ethanol fuel produced (Pimentel and Patzek 2005). The average cost to produce a liter of ethanol from switchgrass feedstock is approximately 54 cents, which is nearly nine cents higher than that for corn grains (Pimentel and Patzek 2005). One of the major cost factors in converting switchgrass feedstock into bioethanol is that of microbial enzymes, which are used to hydrolyze and break down the lignocellulosic biomass into fermentable sugars that can be used for biofuel production (Ragauskas et al. 2006). Presently, microbial hydrolysis enzymes are manufactured in large industrial bioreactors (Lynd et al. 2008). This process is extremely expensive and consequently, the cost of enzymes to produce one gallon of ethanol from lignocellulosic feedstock is roughly 30 cents per gallon (Bothast and Schlicher 2005).

In order to combat the high cost of microbial hydrolysis enzymes, current investigations are working towards expressing cell-wall degrading enzymes in important crop species. The most well studied cell-wall degrading enzymes are the cellulases, a family of enzymes that are naturally found in fungi, bacteria, and some animals (Sukumaran et al. 2005). These enzymes hydrolyze cellulose to produce glucose, cellobiose, and cellooligosaccharides (Sukumaran et al. 2005). There are three major types of cellulase enzymes: cellobiohydrolases, endo-1,4-p-glucanases, and P-glucosidases (Sukumaran et al. 2005). All three types of cellulase enzymes work collectively to break down cellulose into glucose monomer subunits that can then be fermented to yield bioethanol.

The best-studied of the cellulase enzymes is endo-1,4-p-glucanase. In 2000, Ziegler et al. inserted the catalytic domain of the endo-1,4-p-D- glucanase E1 gene (subsequently referred to as E1) from Acidothermus cellulolyticus into Arabidopsis and targeted protein localization to the apoplast (Ziegler et al. 2000). The authors were able to obtain levels of recombinant endoglucanase E1 between 0.01 to 25.7 percent of the total soluble protein (TSP). Novel zymogram assays further confirmed that the catalytic endoglucanase domain was biologically active (Ziegler et al.

2000). A similar study was performed in transgenic potato in which the entire endoglucanase E1 gene from A. cellulolyticus was targeted to mature leaves. Full-length recombinant endoglucanase E1 protein accounted for 2.6 percent of TSP in these transgenic potato plants (Dai et al. 2000a), which is an improvement over the 1.3 percent of partial endoglucanase E1 in TSP extracts of tobacco plants that were transformed using the same method (Dai et al. 2000b).

The successful expression and production of cell wall degrading enzymes in model plant species, such as Arabidopsis and tobacco, opened the door for utilizing this strategy in bioenergy crops. In 2007, Oraby et al. inserted the catalytic domain of the endoglucanase E1 gene from A. cellulolyticus into the nuclear genome of embroygenic rice calli via Agrobacterium transformation (Oraby et al. 2007). After regenerating transgenic plants, the E1 enzyme accounted for 2.4 to 4.9 percent of TSP in rice leaves. The presence of E1 also greatly enhanced the conversion of cellulose to glucose in pre-treated transgenic rice straw (Oraby et al. 2007). That same year, Ransom et al. inserted the same partial endoglucanase E1 gene, containing the catalytic domain, into corn embryogenic calluses (Ransom et al. 2007). The construct was placed under control of the cauliflower mosaic virus 35S promoter and introduced via particle bombardment (Ransom et al. 2007). Using this method, the authors were able to obtain up to 1.16 percent of biologically active recombinant endoglucanase E1 in TSP extracts (Ransom et al. 2007).

A recently published study performed by researchers from Agrivida Inc. (Medford, MA) investigated expressing two xylanase genes in maize under the direction of two different promoters (Gray et al. 2011). Xylanases are another family of cell wall degrading enzymes that act in correlation with cellulases to convert hemicellulose and cellulose into fermentable pentose sugars. The xynB gene from Clostridium stercorarium and the bsx gene from Bacillus sp. were cloned and optimized for expression in maize. After removing bacterial secretion signals, each gene was fused to two signal peptides individually: the barley a-amylase signal peptide sequence (BAASS), which targets protein accumulation to the cell wall, or the rice glutelin B-4 signal peptide (GluB4SP), which would allow for kernel-specific expression. The xylanase sequences that were fused to BAASS were placed under control of the constitutive rice rubi3 promoter, whereas the sequences that were fused to GluB4SP were directed by the rice GluB-4 gene promoter. All constructs were inserted into embryogenic calluses by Agrobacterium — mediated transformation. After transformed plants were regenerated, all of the T0 transgenic maize plants that constitutively expressed both xylanase genes displayed severely stunted growth phenotypes. In GluB4SP transgenic plants, where xylanase expression was directed to the seeds, the plants exhibited normal somatic tissue development, however, the corn grains appeared shriveled. Constitutive expression of both xylanase genes resulted in relatively low accumulation of BSX and XYB proteins in corn stover (0.1 percent TSP). Given that transgenic plants were undersized, higher levels of BSX and XYB accumulation may be lethal to the plant. However, seed specific expression of BSX and XYB resulted in up to four and 16.4 percent TSP, respectively. Presently, further research is being conducted to control xylanase activity and expression in an effort to prohibit negative growth phenotypes associated with expression of these genes in maize. In another case, a gene encoding a thermostable GH10 xylanase, Xy110B, from the hyperthermophilic bacterium Thermotoga maritima, was expressed in transplastomic tobacco plants (Kim et al. 2011). The accumulation levels of the enzymatically active Xy110B were between 11 and 15 percent of the total soluble protein in tobacco leaves. The enzyme displayed "exceptional" thermostability and catalytic activities over methylglucuronoxylose (MeGXn), a major form of xylan in woody plants. The enzyme was also biologically active, hydrolyzing MeGXn into fermentable sugars between 40 and 90°C, and was stable in dry and stored leaves. The transplastomic plants, as well as the progenies, appeared morphologically normal. Due to the harsh pretreatments needed for lignocellulosic feedstocks, selection of thermostable and extreme pH tolerant cellulases and xylanases is quite important for the recombinant enzymes to remain active after the pretreatments. Alternatively, one can work with engineers to develop milder pretreatment conditions and choose appropriate enzymes that can survive the best for those conditions. Moreover, the possibility to bypass pretreatment in certain transgenic alfalfa plants has been reported (Chen and Dixon 2007).

A similar strategy could be used to improve switchgrass as a feedstock. Cellulase enzymes need to be added to the switchgrass feedstock during alcohol production in order to hydrolyze cellulose and produce sugars for fermentation. Cellulases normally include endoglucanase, exoglucanase, and cellobiase (Keshwani and Cheng 2009) and the cost of added cellulases to the process is one of the remaining major economical obstacles for commercial alcohol production from lignocellulosic feedstocks. Currently, no reports have investigated expressing cellulase genes in switchgrass, a strategy that would no doubt facilitate saccharification and reduce the production cost.

Harsh pH and high temperature conditions during pretreatment of the feedstock is a major concern for the survival of the introduced enzyme(s). To overcome this problem, the mildest pretreatment, ammonia fiber explosion (AFEX), was applied to E1-transgenic tobacco biomass and roughly one third of the heterologous enzyme activity was retained. Alternatively, to circumvent the pretreatment stage, crude extract of the E1-transgenic rice plants was added to pretreated rice straw or corn stover and approximately 30 and 22 percent of the cellulose in these plants was converted into glucose, respectively (Sticklen 2006). The expression of cellulase genes in these plants did not have an obvious detrimental effect on plant growth and development. Targeting of these genes to cellular compartments could facilitate accumulation of the heterologous enzyme(s). In switchgrass, about 26 percent of the dry weight is hemicellulose (Keshwani and Cheng 2009), which is currently underutilized for fermentable sugar production and has a great potential for biofuel production in the future.

DOE-USDA awarded Agrivida Inc. (Medford, MA) a grant for producing switchgrass with cell wall degrading enzymes that would remain inactive during plant growth but become activated after harvest. Other laboratories are working to create transgenic switchgrass plants expressing endoglucanase (data unpublished). Using the information obtained from previous research in cereal crops (Oraby et al. 2007; Gray et al. 2011), combined with an efficient transformation system (Li and Qu 2011), switchgrass is a promising candidate for producing cell-wall degrading enzymes as a value-added trait. Introducing value-added traits, such as bioplastics and cell wall degrading enzymes, into important bioenergy crops will ultimately combat the high costs associated with turning the lignocellulosic feedstock into biofuels.

Lignin is a phenolic compound composed of three-dimensional amorphous phenylpropane units or C9 units. The most common phenylpropane unit consists of p-courmaryl alcohol, coniferyl alcohol and sinapyl alcohol units (Fig. 1), which are water insoluble. However, the compounds may be significantly dissolved in low-molecular weight organic solvents such as methanol and ethanol (Mantanis 1994). When lignocellulosic compounds are hydrolyzed by strong sulfuric acid, most of the lignin is insoluble in the acid solution and will precipitate as acid-insoluble lignin. However, small quantities of lignin, known as acid-soluble lignin (ASL) remain in solution and may be determined using analytical procedures such as NREL Laboratory Analytical Procedures (NREL LAP-004) to complete the mass balance for total lignin fractions.

Lignin is intermixed with cellulose and hemicellulose polymer chains by intra — and inter-polymer linkages. Lignin polymers are connected to each other mainly through ether bonds and carbon-carbon bonds (Fig. 2). Ether bonds constitute two-thirds of the total bonds between the lignin

monomers. In addition, lignin is connected to cellulose and hemicelluloses via hydrogen bonds and ester bonds (Gupta 2008; Harmsen et al. 2010). These bonds play an important role in selection of pretreatment method to remove lignin from the biomass.

Softwoods contains higher amounts of lignin compared to hardwoods and grasses (Table 1). In hardwoods, lignin is typically composed of guaiacylpropane and syringylpropane, with a small amount of p-hydroxyphenylpropane units (Lee 2005). In softwoods, lignin is typically constituted of guaiacylpropane with traces of p-hydroxyphenylpropane units (Lee 2005). Lignin composition in grasses mainly contains both guaiacylpropane and syringylpropane units with a small amount of p-hydroxyphenylpropane units (Lee 2005).