There is a growing need of substrate flexibility for biobased production of energy and value-added products that allows the application of variable biodegradable residues within a circular economy. It can be used to balance fluctuating energy provision of other renewable sources. Hydrolysis presents one of the biggest limitations during anaerobic digestion. Methods to improve it will result in broader process applicability and improved integration into regional material cycles. Recently, one focus of anaerobic digestion research has been directed to systems with a separate hydrolysis–acidogenesis stage as it might be promised to improve process performance. Conditions can be adjusted to each class of microorganisms individually without harming methanogenic microorganisms. Extensive research of separate biomass pretreatment via biological, chemical, physical or mixed methods has been conducted. Nevertheless, several methods lack economic efficiency, have a high environmental impact or focus on specific substrates. Pretreatment via a separate hydrolysis stage as cell-driven biotransformation in a suspension might be an alternative that enables high yields, flexible feeding and production, and a better process control. In this review, we summarize existing technologies for microbial hydrolytic biotransformation in a separate reactor stage and the impacts of substrate, operational parameters, combined methods and process design as well as remaining challenges.