Quantification of depth-dependent microbial growth in encapsulated systems

Encapsulated systems have been widely used in environmental applications to selectively retain and protect microorganisms. The permeable matrix used for encapsulation, however, limits the accessibility of existing analytical methods to study the behaviour of the encapsulated microorganisms. Here, we present a novel method that overcomes these limitations and enables direct observation and enumeration of encapsulated microbial colonies over a range of spatial and temporal scales. The method involves embedding, cross-sectioning, and analysing the system via fluorescence in situ hybridization and retains the structure of encapsulants and the morphology of encapsulated colonies. The major novelty of this method lies in its ability to distinguish between, and subsequently analyse, multiple microorganisms within a single encapsulation matrix across depth. Our results demonstrated the applicability and repeatability of this method with alginate-encapsulated pure (Nitrosomonas europaea) and enrichment cultures (anammox enrichment). The use of this method can potentially reveal interactions between encapsulated microorganisms and their surrounding matrix, as well as quantitatively validate predictions from mathematical models, thereby advancing our understanding of microbial ecology in encapsulated or even biofilm systems and facilitating the optimization of these systems.