<p>Recently, Bio-fuel has drawn attention as an alternate fuel due to its advantages over fossil fuels. Many researchers have laid stress on the fermentation of biofuels from renewable and sustainable resources. The continuous rise in the cost of petroleum, depleting fossil fuel reserves and increasing environmental pollution have prompted the scientists to explore alternative energy resources. The sustainable methods are therefore required to be used in producing biofuels to handle the problems related to the hike in crude oil prices, global warming, and decreasing fossil fuel reserves. Bio-fuels like butanol can be generated by using different <i>Clostridium</i> species through the ABE (Acetone-Butanol-Ethanol) process. This paper describes challenges of using <i>Clostridium</i> species, including the pretreatment process. Hydrolysis results in the formation of low and side products. In order to produce biobutanol employing different microbiologic species, it would be essential to develop some novel and promising strains that could overcome technological difficulties in terms of effective butanol production at industrial level.</p> Graphical abstract <p></p>