Perennial grasses (lignocellulosic biomass) such as switchgrass (Panicum virgatum), Miscanthus, and Napier grass (Pennisetum purpureum) have been gaining atten­tion recently for use in biofuel production because of

their low energy requirement for production in the US and Europe, and high biomass yield (Khanna et al.,

2008) . Miscanthus species most often used in biomass research is the sterile hybrid Miscanthus x giganteus, a hardy and fast growing C4 grass that is cultivated via rhizomes (Lewandowski et al., 2000). Yields per acre vary depending on where the crop is grown. The typical yield is 4—10 tons/acre, but yields have been known to reach 16 tons/acre in southern Europe (Lewandowski et al., 2000). Recent research on M. x giganteus and switchgrass at the University of Illinois has produced an average yield of 12 tons/acre and a maximum of

24.7 tons/acre for M. x giganteus and approximately 5 tons/acre for switchgrass (Heaton et al., 2008). The harvestable biomass of Miscanthus is 190% greater than that of corn and could produce 742 more gallons of ethanol per acre (Heaton et al., 2008) or 600 more gallons of butanol per acre. Napier grass, which belongs to sug­arcane family and a native to Africa, is now found in most tropical and subtropical regions of the world (Pen — nisetum purpureum, 2013). It has a high moisture content of 70—80% and reaches maturation following 8 months of plantation/rationing. Approximately two-thirds of Napier grass biomass (dry weight) is composed of sugars: glucan (38.43%), xylan (20.20%), galactan (2.02%), arabinan (2.73%) and mannan (0.23%), and lignin accounted for 20.93% of the lignocellulosic mate­rial, with ash (7.75%) and extractives (1.76%) comprising the remaining fraction (Takara and Khanal 2011). Napier grass has a rapid and dense growth, which have attracted the attention of researchers as a potentially ideal source for lignocellulosic biomass. Napier grass is capable of producing 42 dry tons/acre/year, approximately double the biomass yields of sugarcane and switchgrass (McLaughlin and Kszos, 2005; Takara and Khanal, 2011).

Nonetheless, one of the key steps in the lignocellu — losic biomass-to-fermentable sugars conversion is pre­treatment. The goal of pretreatment is to disrupt the biomass structure and disentangle lignin—carbohydrate complex such that enzymatic hydrolysis of the carbohy­drate fraction of the lignocellulosic biomass-to-simple sugars can be achieved more rapidly and with greater yield (Mosier et al., 2005; Ezeji and Blaschek, 2010). Economic analysis of the current pretreatment methods has shown that the relatively high costs of biofuel (ethanol) production from lignocellulosic biomass arise mainly from costs associated with three factors: (a) harsh pretreatment conditions (high temperature, high pres­sure, use of acids or bases, long residence time, and so on, allowing for inhibitor formation); (b) overuse of expensive enzymes; and (c) recovery of end products (low ethanol concentration in beer; Eggeman and Elan — der, 2005; Ezeji and Blaschek, 2010). Technologies that lead to improvement in any of these areas will help to make isobutanol production using energy crops as feedstock more cost-effective. Moreover, energy crops can be genetically modified to improve biomass yield (per acre per year) without the risk of compromising grain yield or quality along with reducing their recalci­trance to efficient deconstruction to monomeric sugars.

While producing microorganisms have not been shown to directly utilize lignocellulosic biomass as a car­bon source for isobutanol production, Higashide et al.

(2011) recently demonstrated the first production of iso­butanol from crystalline cellulose using C. cellulolyticum. This breakthrough was accomplished after a couple of attempts. First, the activities of the first three enzymes in the isobutanol production pathway were examined by transforming plasmids expressing alsS or alsS ilvCD into C. cellulolyticum and no C. cellulolyticum alsS or alsS ilvCD transformants were obtained. Realizing that alsS and alsS ilvCD transformants could not be obtained, a second attempt wherein genes encoding B. subtilis a-acetolactate synthase, E. coli acetohydroxyacid isomer — oreductase, E. coli dihydroxyacid dehydratase, L. lactis KDC, and E. coli and L. lactis ADHs (alsS, ilvCD, kivd, and adhA, complete isobutanol production pathway genes) were expressed in C. cellulolyticum (Higashide et al., 2011). Despite a mutation in alsS, the alsS ilvCD kivd adhA strain produced 140 and 420 mg/l isobutanol from cellobiose and cellulose, respectively. When plas­mids expressing kivd yqhD alsS ilvCD, in which alsS was the third gene in the operon, was constructed and transformed into C. cellulolyticum, 364 and 660 mg/l isobutanol were produced from cellobiose and cellulose, respectively (Table 7.2). Given the fact that isobutanol production technology has been changing at a rapid pace, this accomplishment in which cellulose is used as a carbon source is significant because it opens the frontier for utilizing lignocellulosic biomass such as energy crops for isobutanol production.