The increased demand for energy, regular price hikes of gasoline, geo-political factors, and environmental damage have alarmed the scientific community, economists and governments to evaluate the potential of cellulosic ethanol as a sustainable, economic and ecofriendly alternative of gasoline [1–3]. Sugarcane bagasse (SB) is an excellent raw material for second generation ethanol production in countries like Brazil, India and China where it is generated in huge amount every year . In Brazil, around 163–169 million tons of SB was generated in the 2012/13 harvest . Almost 50% of SB is used for energy generation in industry but the rest remains unused. The judicious conversion of the left over SB into second generation ethanol may have sustainable, economic and strategic benefits; however it needs intensive technological and multidisciplinary efforts . SB is primarily comprised of carbohydrate polymers (hemicellulose and cellulose) and lignin linked into a highly complex and recalcitrant matrix. Because of this, pretreatment is an inevitable process to render the carbohydrate fraction accessible for cellulolytic enzyme action to release fermentable sugars followed by their bioconversion into ethanol by microbial fermentation. Together, pretreatment and saccharification contributes 50-60% of the total cost incurred for bioethanol production from lignocellulosics . In the past, several pretreatment methods such as auto-hydrolysis, liquid hot water, super critical fluids, alkali, acid, organic solvents, and biological pretreatments have been applied to SB  to make it more amenable to saccharification. Despite several developments in pretreatment processes for SB, complete mechanisms involved in biomass deconstruction remain to be elucidated thus complicating the decision of which pretreatment to apply .
Ammonium hydroxide mediated pretreatment is a state-of-the-art technology which specifically acts on the lignin-carbohydrate complex by cleaving ester linkages via ammonolysis to remove lignin and increase the accessibility of the remaining holocellulose fraction to cellulase action . Ammonia pretreatment in any form such as ammonia fiber expansion (AFEX), ammonia freeze explosion and soaking in aqueous ammonia (SAA) has been proven as an effective strategy for the delignification of various lignocellulosic substrates [5–13]. Key for the success of AFEX is selection of influential parameters in their appropriate ranges to maximize lignin removal while minimizing carbohydrate degradation [5, 9]. After pretreatment, the ammonia solution can be recovered for subsequent use as a delignification agent to further economize the process .
The Taguchi method is a statistical method that utilizes the fundamental principles of statistics, randomization and replication for the design and analysis of factorial experiments. This method allows the investigation of the influence of several parameters on a process while saving time by reducing the number of experiments required .
The delignified material can be separately hydrolysed by cellulolytic enzymes and the resultant sugar syrup can be fermented directly into ethanol by an appropriate microorganism (SHF). Another option is to perform the enzymatic hydrolysis and fermentation of all released sugars (pentose and hexose) into ethanol simultaneously (SSF). The latter has shown several advantages such as process intensification, less processing time, low contamination risk, cost reduction and high ethanol titers . The most important feature of SSF is to overcome the enzyme inhibition by the released sugars during enzymatic hydrolysis as the released sugars are simultaneously converted into ethanol by the microorganism. However, the difference in optimum temperature for microbial fermentation and enzymatic hydrolysis is a major concern of SSF [3, 15].
Here, we report the ammonia pretreatment optimization of SB and the efficiency of all pretreated bagasse samples for sugar recovery during enzymatic hydrolysis. Ethanol production was evaluated under SHF and SSF using the sugar hydrolysate of optimized ammonia pretreated bagasse or delignified SB by S. stipitis NRRL Y-7124. Structural and crystallinity changes in native, ammonia pretreated and enzymatically digested SB were also analyzed by SEM, XRD and solid-state 13C NMR spectroscopy.