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Bioethanol is the most commonly used biofuel. It is an alternative to replace fossil fuels in renewable energy; it can be produced from lignocellulosic feedstock using a biotechnological process. Their participation of microorganisms is crucial in the bioconversion process of fermentation for ethanol production and can involve bacteria, fungi, and yeasts. However, when working within bioethanol processes from lignocellulose feedstock, microorganisms face some challenges, such as high temperature, high solids content, and the ability to ferment sugars for high ethanol concentration. Such challenges will depend on operative strategies, such as simultaneous saccharification and fermentation, separate hydrolysis and fermentation, semi-simultaneous saccharification and fermentation, and consolidated bioprocessing; these are the most common configurations. This review presents different trends of the microbial role, biochemical application, and fermentation operative strategies for bioethanol production of the second generation.