AFEX pretreatment (Dale 1986) is an alkaline pretreatment process that alters physicochemi­cal structure of lignocellulosic biomass at moderate temperatures (60-100°C) and high pres­sures (250-300psi). Like the alkali pretreatment, AFEX pretreatment results in increased wetting of the treated biomass (Sulbaran de Ferrer et al. 1997; Shishir et al. 2007), decrystal­lization of cellulose (Gollapalli et al. 2002), partial depolymerization of hemicellulose, cleav­age of lignin-carbohydrate complex (LCC) linkages (Shishir et al. 2007), and increase in surface area due to structural disruption.

In a typical AFEX pretreatment process using corn stover (Shishir et al. 2007), corn stover is adjusted to 60% moisture (kilogram water/kilogram dry biomass) before being transferred to a high-pressure Parr reactor, and liquid ammonia (1 kg of ammonia/kg of dry biomass) is slowly added to the reactor. The temperature is raised and maintained at 90°C for a period of 5 minutes. Following a 5)minute residence time, the pressure is quickly released. The sudden drop in reactor pressure results in fast vaporization of ammonia, causing an explosive decompression and disruption of the biomass. The pretreated corn stover is stored under a fume hood overnight, during which the residual ammonia is vaporized. Subsequently, the AFEX pretreated corn stover is either subjected to enzymatic hydrolysis or stored in a freezer until further use.

The AFEX pretreatment process has some unique characteristics that distinguish it from other pretreatment methods and they are as follows (Teymouri et al. 2005): (1) a significant amount of the ammonia used in the pretreatment is recovered and reused; (2) AFEX is basically a dry to dry process because there is no waste or liquid stream from the process; (3) pretreated biomass is stable for long periods and can be used with great solid loadings in enzymatic hydrolysis and fermentation processes; (4) AFEX process yields intact cel­lulose and hemicellulose polymers with little or no degradation; (5) no need for neutraliza­tion prior to the enzymatic hydrolysis of AFEX-pretreated biomass. The AFEX pretreatment process was used recently to pretreat DDGS, and subsequently fermented into ABE (Ezeji and Blaschek 2008a). In this investigation, various Clostridium species were tested for the ability to ferment AFEX-pretreated DDGS to ABE. The AFEX pretreated DDGS (15% total solids loading) generated 41.4g/L total sugars upon enzymatic hydrolysis comparable to that generated by hot water pretreated samples.