Figure 2. (a) Size and (b) count of detected AOB colonies in
three consecutive cross-sections (A1, A2, and A3) from the same alginate
bead after a 5-day batch incubation. The sets of cross-sections with
p-values <0.05 from a Kruskal-Wallis test are marked with
asterisks. Depths with only one
sample are marked with carets and excluded from a Kruskal-Wallis test.
Error bars indicate the 95% confidence intervals. Here a depth of 0 and
3000 represent the outer surface of the bead, with 1500 µm equaling the
center/core of the bead.
Encapsulated growth of Nitrosomonas colonies
Initial bead samples and bead samples taken after one day of incubation
were analyzed for bacterial numbers and colony area (Figure S7). Under
aerobic batch incubation in triplicate reactors, the encapsulatedNitrosomonas consumed ammonium at 42 ± 9 mgN/L-d and produced
nitrite at 39 ± 13 mgN/L-d (n = 3). Encapsulated AOB colonies were
initially distributed evenly throughout the depth range of 0 to 1500 µm
(bead core) with an average colony size of 20 µm2 and
an average density of 1,360 colonies mm-3. Such AOB
distribution was expected as the alginate and bacterial culture were
homogenized before crosslinking. No significant change in distribution
was observed after the 1-day incubation of encapsulated AOB under
pairwise comparison (Wilcoxon test, p>0.05). This is also
expected, because the doubling time of encapsulated Nitrosomonasis around 0.5 d ; therefore, one day of incubation is likely to be
insufficient to develop clear differences in colony morphology and
number.
The same experimental setup was used to incubate encapsulatedNitrosomonas for 5 days. Three different beads were collected and
cross-sectioned at the end of the incubation. Three consecutive slices
(at depth D ± 10 µm, where D is every 100 µm until 1200
µm) from the same bead were analyzed and used as replicates to represent
the colony profile at each depth D . As observed previously, the
initial AOB numbers and colony sizes were evenly distributed through the
bead depth of 0 to 1200 µm (Figure S7). Ammonium consumption and nitrite
production was also similar to that previously observed (Figure S2). In
contrast to the 1-day incubation, however, a different spatial
distribution of encapsulated AOB colonies was observed after the 5-day
incubation, as plenty of time had been given for the microorganisms to
grow (Figure 3 ). Preferential growth of encapsulated colonies
was detected near the surface (300-500 µm), shown by increased size and
density of AOB colonies at those depths. Some variations existed between
the depth profiles of AOB colonies obtained from different bead samples,
but overall, the trends were observable, quantifiable, and reproducible.
Of note is the fact that the data in Figure 3 represents triplicate
slices in three separate beads. Size of colonies were statistically
different from each other in the three beads (Kruskal-Wallis test, p
< 0.05), however, the colony counts were not significantly
different among the three beads (Kruskal-Wallis test, p >
0.05).