Figure 3. The distribution of (a) size and (b) count of AOB colonies detected by FISH across depth in three different alginate beads (A, B, and C) after a 5-day batch incubation. Error bars indicate the 95% confidence intervals.
In contrast to our microtome cross-sectioning and FISH method, qPCR could only provide average results for an entire bead, and as a result, could not provide information on changes in the spatial distribution of encapsulated colonies over time (Figure 4a ). Since DNA was extracted after the dissolution of the entire alginate bead, the qPCR results served as an average count across the depth profile for all encapsulated cells. No significant change was observed in either the 16S rRNA gene or amoA copy numbers after the 1-day incubation (p<0.05, student t-test), which agrees with the results from cross-sectioning and FISH methods (Figure S7). In contrast, significant increases in copy number were observed for both genes after the 5-day incubation (p<0.05, student t-test). Increase in the copy number amoA again agrees with the cross-section FISH results showing the growth of AOB colonies near the bead surface (Figure 3 ). The difference between increases in the 16S rRNA gene andamoA copy numbers could be attributed to the potential growth of heterotrophs within and on the surface of alginate beads. These heterotrophs may have grown by using carbon fixed and secreted by AOB and/or by degrading alginate . The cross-sectioning and FISH method presented herein, however, provides much more detailed data and enables a much clearer understanding of growth in an encapsulated system. The details of these results also facilitate the validation of mathematical models (e.g., Wang et al., 2022).