Although the Iogen process relies on acid pretreatment and cellulase digestion, Dan­ish investigators rank other pretreatment methods (with short residence times, 5-6 minutes) as superior for subsequent wheat straw cellulose digestion by cellulase (24 hours at 50°C):8

Steam explosion (215°C) > H2O2 (190°C) > water (190°C) > ammonia (195°C) > acid (190°C)

The degradation of cellulose to soluble sugars was enhanced by adding nonionic surfactants and polyethylene glycol during enzymatic hydrolysis; the best results were obtained with a long-chain alcohol ethoxylate in conjunction with steam explo — sion-pretreated wheat straw, and the additives may either have occupied cellulase binding sites on residual lignin or helped to stabilize the enzyme during the lengthy digestion.8

In addition, the attention of Novozymes (one of the world’s major enzyme pro­ducers) has evidently been attracted post-2004 by wheat straw and the problems of its complete conversion to fermentable sugars:

• Arabinoxylans form an undigested fraction in the “vinasse” (the insoluble fermentation residue) after the end of a wheat-based bioethanol process; a mixture of depolymerizing enzymes from Hypocrea jecorina and Humi — cola insolens could solubilize the insoluble material and release arabinose and xylose — although at different rates with different optimal pH values and temperature ranges for the digestion.9

• A subsequent study mixed three novel a-L-arabinofuranosidases with an endoxylanase and a P-xylosidase to liberate pentoses from water-soluble and water-insoluble arabinoxylans and vinasse; much lower enzyme activi­ties were required than previously, and this may be a technology for pentose release before wheat straw-substrate fermentations.10

• Mixtures of a-L-arabinofuranosidases from H. insolens, the white-rot basidomycete Meripilus giganteus, and a Bifidobacterium species were highly effective in digesting wheat arabinoxylan, the different enzymes acting synergistically on different carbohydrate bonds in the hemicellulose structures.11

Arabinans constitute only 3.8% (by weight) of the total carbohydrate (cellulose, starch, xylose, and arabinose) in wheat straw, and a lack of utilization of all the pentose sugars represents a minor inefficiency. Releasing all the xylose (as a sub­strate for the engineered xylose-utilizing yeast) — xylose constitutes 24% of the total sugars — and completing the depolymerization of cellulose to (insofar as is possible) free glucose are more significant targets for process improvement.

As in Canada, wheat straw has been assessed to be a major lignocellulosic feed­stock in Denmark.[33] Substrate pretreatment studies from Denmark have, unlike at Iogen, concentrated on wet oxidation, that is, heat, water, and high-pressure O2 to hydrolyze hemicellulose while leaving much of the lignin and cellulose insoluble; vari­ous conditions have been explored, including the combining of thermal hydrolysis, wet oxidation, and steam explosion.12-15 The major concern with this type of pretreatment method is — with so many biomass substrates — the formation of inhibitors that are toxic to ethanologens and/or reduce ethanol yield.16-18 These degradation products of lignocellulosic and hemicellulosic polymers include aromatic acids and aldehydes as well as aliphatic carboxylic acids and sugar-derived components; laboratory strains of Saccharomyces cerevisiae exhibit differential responses to the growth inhibitors, and cell-free enzyme preparations with cellulase and xylanase activities were severely inhibited by chemically defined mixtures of the known wheat straw inhibitors, with formic acid being by far the most potent inhibitor.19 The positives to be drawn were, however, that even a laboratory strain could grow in 60% (w/v) of the wheat straw sub­strate and that a focused removal of one (or a few) inhibitors (in particular, formic acid) may suffice to render the material entirely digestible by engineered yeast strains.