Complete microbial encapsulation within alginate-εPLL core-shell hydrogel beads enables spatially distributed co-cultures and bioreactor scale-up

Achieving stable and reproducible microbial co-cultures requires tools to control the population balance among species. To address this challenge, here we present an effective method for microbial encapsulation relying on alginate-core hydrogel beads coated with an alternating ε-poly- l -lysine/alginate multilayer shell (εPLL-HB). This procedure combines two features that are rarely reported together: (i) the complete containment of several microbial species (prokaryotic and eukaryotic) under diverse conditions, while (ii) supporting their controlled growth inside the εPLL-HB. The lack of cell leakage from the capsules is often overlooked and has only been achieved with shells prepared by polymerization reactions. In contrast, εPLL-HB manufacturing is simpler and biocompatible, only relying on non-covalent interactions. In this regard, εPLL showed better performance than chitosan and α-poly- l -lysine, two of the biomaterials most used as coating agents in core-shell encapsulation. Therefore, εPLL-HB allowed us to build spatially distributed co-cultures, effectively balancing populations of microorganisms with different growth rates and interactions. We also demonstrate that εPLL-HB are scalable to stirred-tank bioreactor cultures, proving their utility in large-scale applications. Furthermore, microbial-loaded εPLL-HB maintained their encapsulation efficacy and cell viability after long-term storage at −80 °C and provided protection against toxic compounds in lignocellulosic-derived media. The superior microbial containment, scalability, structural integrity, and chemical resistance of εPLL-HB, combined with their cost-effective and simple preparation, make them a versatile tool for engineering synthetic microbial consortia, with broad applicability in biotechnological processes. • εPLL outperforms other polymers when used as coating agent of hydrogel beads ( HB). • εPLL-HB show a complete containment of diverse microbial cells while sustaining their growth. • εPLL-HB enable the construction of spatially distributed co-cultures. • εPLL-HB maintain their performance when used in scaled-up cultures in a bioreactor.