Corn stover is the above-ground plant from which the corn grain has been removed, and the constituent parts are leaves, stalk, tassel, corn cob, and shuck (the husk around the grains when in the intact cob); up to 30% by dry weight of the harvested plant is repre­sented by the collected grain. In one of the earliest technological and economic reviews of corn-based fuel alcohol production, corn stover was included for consideration—but solely as an alternative to coal as a boiler fuel for distillation. In late 1978, the report for the U. S. DOE estimated that corn stover would increase the final cost of fuel ethanol by 40/gallon as the use of corn stover as a fuel entailed costs roughly double those of local Illinois coal.36 The use of corn stover was, therefore, considered to be “justified only if the plant is located in an area where transportation cost would cause a doubling of the coal cost, or environmental considerations would rule against the use of coal; neither of which is very likely.” Such arguments left corn stover in the field as an aid against soil erosion for over a decade until the option of lignocellulosic ethanol began to be seriously considered. By 2003, the National Renewable Energy Laboratory, Golden, Colorado, estimated the annual and sustainable production of corn stover as 80-100 million dry tonnes/year, of which 20% might be utilized in the manufacture of “fiber” products and fine chemicals (e. g., furfural); 60-80 million dry tonnes would remain as a substrate for bioethanol production.37 Five years earlier, an estimate of total corn stover availability had been more than 250 million tonnes, with 30 million being left on the fields for erosion control, leaving 100 million available for biofuels production.38 With expand­ing corn acreage and a definite future for corn-based ethanol, a supply of corn stover is ensured — and commercial drivers may direct that starch ethanol and “bioethanol” facilities might be best sited adjacent to one another (see chapter 2, section 2.6).

With corn stover rising up the rankings of biomass substrates for ethanol produc­tion in the United States and elsewhere, experimental investigations of pretreatment technologies has proliferated since 2002.39-50 From this impressive corpus of practi­cal knowledge, some reinforced conclusions are apparent: [34]

temperatures (25-55°C), the enzymic digestibility of the resulting cellulose was highly influenced by both the removal of acetylated hemicellulose residues and delignification, but deacetylation was not seriously influenced by the levels of O2 or the temperature.58 Adding a water washing to ammonia-pretreated material removed lignophenolic extractives and enhanced cellulose digestibility.59 Grinding into smaller particles increased the cellulose digestibility after ammonia fiber explosion, but the chemical compositions of the different particle size classes showed major changes in the contents of xylans and low-molecular-weight compounds (figure 4.4). This could be explained by the various fractions of corn stover being differentially degraded in smaller or larger particles on grinding; for example, the cobs are rela­tively refractive to size reduction; the smaller particle sizes after AFEX treatment were more cellulase-degradable than were larger particles. Electron microscopic chemical analysis of the surface of the pretreated material provided evidence that lignin-carbohydrate complexes (chapter 2, section 2.3.2) had been disrupted.59 The high hemicellulose content of corn cobs has been exploited in a development where aqueous ethanol-pretreated material is washed and then hydrolyzed with an endox — ylanase; food-grade xylooligosaccharides can easily be purified, and the cellulosic material is readily digestible with cellulase.60 An additional advantage of corn cobs is that they can be packed at high density, thus reducing the required water inputs and giving a high concentration in the xylan product stream.

The dominance of inorganic acids for acid pretreatment of biomass substrates has only recently been challenged by the use of maleic acid, one of the strongest organic dicarboxylic acids and a potential mimic of the active sites of hydrolase enzymes with two adjacent carboxylic acid residues at their active sites.61 In com­parison with dilute sulfuric acid, maleic acid use resulted in a greatly reduced loss of xylose at high solids loadings (150-200 g dry stover/l), resulting in 95% xylose

yields, only traces of furfural, and unconditioned hydrolysates that could be used by recombinant yeast for ethanol production; 90% of the maximum glucose release could be achieved by cellulase digestion of the pretreated stover within 160 hours.

Examination of (and experiments with) the cellulase digestion of pretreated corn stover have led to other conclusions for industrial applications:

• Studies of the binding of cellobiohydrolase to pretreated corn stover identi­fied access to the cellulose in cell wall fragments and the crystallinity of the cellulose microfibrils after pretreatment to be crucial.62

• Adding small amounts of surfactant-emulsifiers during cellulase digestion of pretreated corn stover also increased the conversion of cellulose, xylan, and total polysaccharide to sugars, by acting to disrupt lignocellulose, sta­bilize the enzyme, and improve the absorption of the enzyme to the mac­roscopic substrates.63

• With steam-pretreated corn stover, near-theoretical glucose yields could be achieved by combining xylanases with cellulase to degrade residual hemi — cellulose bound to lignocellulosic components.64

• The initial rate of cellulase catalyzed hydrolysis is influenced strongly by the cellulose crystallinity whereas the extent of cellulose digestion is most influ­enced by the residual lignin.65 Modern methods of polymer analysis (e. g., diffusive reflectance infrared and fluorescence techniques) used in this work may be adaptable to on-site monitoring of pretreated biomass substrates.

• The formation of glucose from pretreated corn stover catalyzed by cellulase is subject to product inhibition, and the effects of substrate concentration and the amount (“loading”) of the enzyme are important in determining kinetic parameters.66

• Cellulase and cellobiohydrolase can both be effectively recovered from pre­treated and digested corn stover and recycled with consequent cost savings of approximately 15% (50% if a 90% enzyme recovery could be achieved).67

• The solid material used for cellulase-catalyzed hydrolysis itself is a source of potential toxic compounds produced during pretreatment but trapped in the bulk solids; activated carbon is (as discussed above) effective in removing acidic inhibitors from the liquid phase resulting from digestion of the reintroduced substrate.68

A comparative study of several methods for corn stover pretreatment concluded that alkaline methodologies had the potential to reduce the quantities of cellulase necessary in cellulose digestion but that hemicellulase activities may require supplementation.69