An integrated bioconversion process, which incorporated soaking in aqueous ammonia (SAA) pretreatment and two-phase simultaneous saccharification and fermentation (TPSSF), was investigated. The TPSSF process consists of pentose conversion using recombinant Escherichia coli KO11 in the first phase (0-48h) and hexose conversion with Saccharomyces cerevisiae D₅A in the second phase (48-96h). With the xylan-rich SAA-pretreated corn stover as substrate, the TPSSF process resulted in 84% of theoretical maximum ethanol yield based on the total sugars (glucan+xylan) in untreated corn stover.

Cascade two-phase simultaneous saccharification and fermentation (CTPSSF) was also studied to improve overall efficiency of enzymes. SAA-pretreated corn stover was subjected to two-phase SSF performed in series. The enzymes in liquid fraction were recycled from each stage for the use in the subsequent fermentation stage by separating the solid residues from fermentation broth. The results showed that approximately 60% of theoretical maximum ethanol yield based on the total sugars in untreated corn stover was achieved, while enzyme loadings were significantly reduced (up to 50%).

A low-liquid pretreatment method of corn stover with aqueous ammonia was evaluated for the purpose of ethanol production. The effects of a variety of factors, i.e., ammonia loading, solid-to-liquid ratio and reaction time, on the composition and enzyme digestibility of corn stover were thoroughly investigated. Optimal low-liquid ammonia pretreatment conditions (50 wt. % of ammonia loading, 1:5 of solid-to-liquid ratio, 30°C, 4 weeks) resulted in up to 55% delignification and 86.5% glucan digestibility with 15 FPU/g-glucan of cellulase and 30 CBU/g-glucan of β-glucosidase. With the corn stover treated under the conditions of 50 wt. % ammonia loading, 1:2 solid-to-liquid ratio, 30°C and 4 weeks as the substrate, an ethanol yield of 73% of theoretical maximum was obtained on the basis of the total carbohydrates (glucan+xylan) content in untreated corn stover. The results demonstrated that the pretreatment of corn stover could be achieved with reduced ammonia loading and water consumption compared to those for soaking in aqueous ammonia (SAA) pretreatment. By applying the mild reaction conditions and low ammonia and moisture dosage, the low-liquid aqueous ammonia pretreatment reduces the pretreatment severity and liquid throughput, and has the potential of making the ethanol production process more cost-effective.