Bio-butanol Production from Hydrolysates of Napier Grass Using Clostridium acetobutylicum: A Comparison of Separate and Semi-Simultaneous Fermentation Processes

Abstract

The quest for sustainable and economically viable biofuels has intensified the focus on lignocellulosic biomass as a renewable feedstock. This study aimed to investigate the valorization of Ethiopian Napier grass (Pennisetum purpureum) for biobutanol production via Acetone-Butanol-Ethanol (ABE) fermentation. The biomass was characterized, subjected to optimized alkaline pretreatment (2.5 wt% NaOH, 1.5 wt% urea, 121°C, 40 min), and enzymatically hydrolyzed. Fermentation using Clostridium acetobutylicum was optimized via Response Surface Methodology (RSM) with a Box-Behnken Design (BBD), and two bioprocessing strategies Separate Hydrolysis and Fermentation (SHF) and Semi-Simultaneous Saccharification and Fermentation (sSSF)—were compared. Compositional analysis revealed that Napier grass contains 35.2% cellulose, 16.9% hemicellulose, and 22.4% lignin, offering 52.1% total fermentable carbohydrates. Alkaline pretreatment effectively delignified the biomass, yielding 28.74 g/L of glucose upon enzymatic hydrolysis. The optimized SHF process (37.5°C, pH 6.0, 64.7 h, 6.5% w/w substrate) produced 15.65 g/L butanol with 0.077 g L⁻¹ h⁻¹ productivity and 29.5% efficiency. Notably, the sSSF strategy achieved superior results: 17.6 g/L butanol, 0.083 g L⁻¹ h⁻¹ productivity, and 33.6% efficiency. The distilled biobutanol exhibited favorable fuel properties, including a Higher Heating Value (HHV) of 33,050 kJ/L. This study conclusively demonstrates that Ethiopian Napier grass is a promising feedstock for second-generation biobutanol, with the sSSF strategy offering a more efficient and integrated bioprocessing approach.