Background <p>Biobutanol, a promising biofuel, can be produced through the fermentation of various carbon sources, particularly non-cereal agricultural waste. The aim is to optimise the pretreatment process to maximise butanol accumulation.</p> Methods <p>The work was conducted using butanol strain <i>Clostridium</i> sp. UCM B-7570 and non-graine substrates for cultivation. A gas chromatograph was used to determine the present of ethanol, acetone, and butanol in the culture liquid.</p> Results <p>We optimised the technological parameters of butanol production from lignocellulosic biomass, emphasising the performance indicators crucial for developing commercially viable technology and industrial-scale production. Novel domestic lignolytic bacterial strain was isolated and identified. This strain holds promise for enhancing enzymatic hydrolysis and subsequent utilisation of lignocellulosic components. Co-cultivation with an aerobic partner demonstrated increased butanol accumulation. We investigated various hydrolysis methods using miscanthus as a model substrate. Notably, the study on ultrasonic disintegration of lignocellulosic materials revealed that optimising pretreatment is crucial, and the most effective approach may involve a combination of different hydrolysis techniques depending on the specific biomass type.</p> Conclusion <p>These findings pave the way for significant advancements in biobutanol production technology. They provide a strong foundation for both optimising existing methods and developing entirely new approaches for efficient butanol production.</p> Graphical Abstract <p></p>