Updated Protocol for the Assembly and Use of the Minibioreactor Array (MBRA).

Abstract

The human microbiome comprises diverse and dynamic microbial communities that play essential roles in host health. Understanding these communities and their responses to environmental factors is critical for advancing microbiome-based therapeutics. Traditional in vitro models for cultivating human-derived microbiota often lack scalability and require extensive technical expertise, limiting their accessibility and throughput. To address these limitations, we developed the Minibioreactor Array (MBRA) system -- a modular, single-stage, continuous-flow platform for high-throughput cultivation of microbial communities. This system enables parallel cultivation of up to 48 distinct microbial communities, supporting experimental flexibility while maintaining the stable growth of complex ecosystems. This protocol provides detailed guidance on MBRA fabrication, assembly, sterilization, and operation. The system's modular design allows for easy integration into anaerobic chambers and supports customization for a wide range of experimental applications. It has been used to study microbial responses to antibiotics, dietary compounds, and pathogen invasion, and to screen for pathogen-resistant communities. With its accessibility, scalability, and reproducibility, the MBRA represents a powerful model system for investigating microbial interactions and advancing microbiome research.