In biofuel production via fermentation, SHF is the most conventional and widely used method. The SSF was another method used for conversion of sugars to avoid inhibitory effect occurs in SHF method. Specifically, the SHF method suffers from slow sugar uptake rates due to the feedback inhibition caused by an accumulation of sugars after enzymatic hydrolysis. To assess the effectiveness of our proposed pretreatment procedure, we conducted biobutanol Ionomycin experiments using our hydrolysates as the substrate. We attempted to compare the fontanels ABE production from hydrolysate of sugarcane bagasse by using SSF or SHF methods. An additional enzymatic saccharification pretreatment step (Step 6) was optimized and used to maximize reducing sugar yields before SHF was performed on the collected sugarcane bagasse hydrolysates from Step 2 through Step 6. Alternatively, SSF was performed on the collected hydrolysates from Step 2 through Step 4 as well as the solid–liquid mixture from Step 5 ( Fig. 2).