The widely quoted assessment of softwoods is that, as a biomass substrate, the lig- nocellulose is too highly lignified and difficult to process to yield cellulose easily digested by cellulase — in practical terms, excess enzyme is required and imposes unrealistically high costs and protracted digestion times.170 Nevertheless, the mas­sive resources of softwood trees in the Pacific Northwest of the United States, Can­ada, Scandinavia, northern Europe, and large tracts of Russia maintain softwoods as an attractive potential biomass for fuel alcohol production. Sweden has a particular stake in maximizing the efficiency of ethanol production from softwoods on account of the planned diversion of large amounts of woody biomass to direct heat and power facilities with the phasing out of nuclear generating capacity.70 Much of the published work on softwood utilization for bioethanol indeed derives from Canadian and Scan­dinavian universities and research centers.

Although softwoods are low in xylans in comparison with other biomass crops (table 1.5), their content of glucan polymers is high; the requirement for xylose­utilizing ethanologens remains a distinct priority, whereas mannose levels are high and should contribute to the pool of easily utilized hexoses. To make the potential supply of fermentable sugars fully accessible to yeasts and bacteria for fermentation, attention has been focused on steam explosive pretreatments with or without acid catalysts (SO2 or sulfuric acid) since the 1980s; pretreatment yields a mixed pentose and hexose stream with 50-80% of the total hemicellulose sugars and 10-35% of the total glucose, whereas a subsequent cellulase digestion liberates a further 30-60% of the theoretical total glucose.70 Because only a fraction of the total glucose may be recoverable by such technologies, a more elaborate design has been explored in which a first stage is run at lower temperature for hemicellulose hydrolysis, whereas a second stage is operated at a higher temperature (with a shorter or longer heating time and with the same, higher, or lower concentration of acid catalyst) to liberate glucose from cellulose.71 Such a two-stage process results in a sugar stream (before enzyme digestion) higher in glucose and hemicellulose pentoses and hexoses but with much reduced degradation of the hemicellulose sugars and no higher levels of potential growth inhibitors such as acetic acid (figure 4.5).

Two-stage pretreatment suffers from requiring more elaborate hardware and a more complex process management; in addition, attempting to dewater sulfuric acid- impregnated wood chips before steaming decreases the hemicellulose sugar yield from the first step and the glucose yield after the second, higher-temperature stage.72 Such pressing alters the wood structure and porosity, causing uneven heat and mass transfer during steaming. Partial air drying appears to be a more suitable substrate for the dual-stage acid-catalyzed steam pretreatment.

Not only is the sugar monomeric sugar yield higher with the two-stage hydroly­sis process, the cellulosic material remaining requires only 50% of the cellulase for subsequent digestion.73 This is an important consideration because steam-pretreated softwood exhibits no evident saturation with added cellulase even at extremely high enzyme loadings that still cannot ensure quantitative conversion of cellulose to sol­uble sugars: with steam-pretreated softwood material, even lavish amounts of cel- lulase can only liberate 85% of the glucan polymer at nonviably low ratios of solid material to total digestion volume, and although high temperatures (up to 52°C) and high agitation rates are helpful, enzyme inactivation is accelerated by faster mixing speeds.74 The residual lignin left after steam pretreatment probably restricts cellulase attack and degradation by forming a physical barrier restricting access and by bind­ing the enzyme nonproductively; extraction with cold dilute NaOH reduces the lignin content and greatly increases the cellulose to glucose conversion, the alkali possibly removing a fraction of the lignin with a high affinity for the cellulase protein.75

In addition to the engineering issues, the two-stage technology has two serious economic limitations:

1. Hemicellulose sugar recovery is aided after the first step by washing the slurry with water but the amount of water used is a significant cost factor for ethanol production; to balance high sugar recovery and low water usage, a continuous countercurrent screw extractor was developed by the National Renewable Energy Laboratory that could accept low liquid to insoluble sol­ids ratios.76

2. The lignin recovered after steam explosion has a low product value on account of its unsuitable physicochemical properties; organic solvent extraction of lignin produces a higher value coproduct.77

As with other biomass substrates, heat pretreatment at extremes of pH gener­ates inhibitors of microbial ethanol production and, before this, cellulase enzyme action.7879 A number of detoxification methods have been proposed, but simply adjusting the pH to 10 to precipitate low-molecular-weight sugar and lignin deg­radation products is effective.80 Six species of yeasts — including S. cerevisiae, Candida shehatae, and species found in forest underbrush in the western United States — were tested for adaptation to softwood (Douglas fir) pretreated with dilute acid, and isolates were selected and gradually “hardened” to hydrolysate toxicity for improved ethanol production.81

Aqueous ethanol pretreatment of softwoods (the lignol process) has been strongly advocated on account of its ability to yield highly digestible cellulose as well as lignin, hemicellulose, and furfural product streams — the extraction is operated at acid pH at 185-198°C, and some sugar degradation does inevitably occur.82 After process optimization, a set of conditions (180°C, 60 minutes of treatment time, 1.25% v/v sulfuric acid, and 60% v/v ethanol) yielded83

• A solids fraction containing 88% of the cellulose present in the untreated wood chips

• Glucose and oligosaccharides equivalent to 85% of the cellulosic glucose was released by cellulase treatment (48 hours) of the treated lignocellulose

• Approximately 50% of the total xylose recovered from the solubilized fraction

• More than 70% of the lignin solubilized in a form potentially suitable for industrial use in the manufacture of adhesives and biodegradable polymers