1 file, Adaptive laboratory evolution (ALE) is a powerful tool for understanding and controlling the evolutionary trajectories of microorganisms. The scope of applications extends widely, including areas such as: biotechnology, synthetic biology, microbial ecology, and fundamental evolutionary research. In this work, we systematically explore the implementation and advantages of mini-bioreactors, defined as reactors with working volumes below 0.5 L, in ALE experiments. Mini-bioreactors offer substantial improvements over traditional large-scale reactors, including: reduced costs, enhanced parallelization capabilities, customizable configurations, and ease of automation. Through the utilization of illustrative case studies, which facilitate a comparative and critical evaluation of: batch, chemostat, turbidostat, and morbidostat operational modes, this review underscores the distinct capabilities of mini-bioreactors in enabling precise, dynamic control of evolutionary pressures. The novelty of this review lies in its comprehensive synthesis of recent advancements in mini-bioreactor technologies and operational strategies, particularly emphasizing innovations, such as: integrated automation, advanced sensors, and novel control algorithms adapted or specially designed for ALE. The ultimate objective is to provide both novices and experienced researchers with an updated, in-depth resource that addresses current technological limitations and future directions of mini-bioreactors in ALE.

Previous literature reviews have discussed ALE and mini-bioreactors separately but have not explored in detail the use of mini-bioreactors in the context of ALE. Earlier works either partially addressed the topic or focused narrowly on specific aspects, missing the breadth of mini-bioreactor applications in ALE. In addition, recent advancements in the design and use of mini-bioreactors for ALE experiments emphasize the need for a comprehensive review. This article provides an up-to-date overview of mini-bioreactors in ALE, discussing their fundamentals, characteristics, modes of operation, and future perspectives, with the aim of filling a significant knowledge gap in the field, This work received funding from PID2022-136817OB-I00, AEI/10.13039/501100011033/FEDER, UE and Xunta de Galicia IN606A-2020/028 and IN607B 2021/11, Peer reviewed