Abstract
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 behavior 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 analyzing
the system via fluorescence in situ hybridization, while
retaining 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 analyze 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.