2.3 Image processing and data analysis
FISH images of cross-sections were obtained using an Olympus IX-81
inverted microscope equipped with a long working distance (WD 2.7-4.0)
40x objective lens (NA-0.6) and a DP73 camera (Olympus, Japan). Image
processing was carried out on the Fiji software (v 2.10.0) . Rolling
ball background subtraction (30 pixels) and particle size
(>2x2 pixels) and circularity (>0.1) filters
were used to correct for uneven background and filter out false
positives from the textures left on the slices by the microtome slicing.
The size and count of fluorescent colonies from each section were
recorded. Colony counts were normalized to the volume of detection, as
calculated by the area of each image (1.47 mm2) and
the thickness of the cross-section (10 μm). Box plots were constructed
in R with a median represented by a centerline, a 25th to 75th
interpercentile range represented by a shaded box, and smallest/largest
values within the 1.5 times interquartile range represented by
upper/lower error bars. Statistical analyses were performed in R (v
4.2.2). Non-parametric tests were used to compare particle sizes. The
Kruskal-Wallis one-way ANOVA was conducted on triplicates at each depth,
whereas the Wilcoxon signed-rank test was conducted between paired
samples before and after each incubation. Student t-tests were performed
on qPCR results to compare gene copy numbers before and after
incubation.
2.4 DNA extraction and qPCR
To extract DNA, an entire alginate bead was dissolved in 1 mL of sodium
citrate (55 mM) and EDTA (30 mM) solution. The dissolved bead solution
was centrifuged at 4 °C to obtain cell pellets, which were washed twice
and resuspended in buffer solution (10 mM Tris, 0.1 mM EDTA) for
storage at –20 °C. The PowerSoil DNA extraction kit (Qiagen) was used
to extract DNA from the pellet in the buffer. qPCR was used to quantify
total bacteria, AOB, anammox bacteria, and NOB with 341F and 805R
primers, amoA_1F and amoA_2R primers, hzsA_1597F and hzsA_1857R
primers, and nxrB169f and mxrB638r primers, respectively (Table S1). The
qPCR was performed on a StepOnePlus Real-Time PCR System (Thermo Fisher)
with the standard curve method as described previously . The
amplification efficiencies for all primer sets were >90%,
except for the 16S rRNA gene (>85%).
Results and Discussion
Method validation with encapsulated Nitrosomonas
The background noise of FISH probes and autofluorescence of alginate
embedded in GMA were first examined using abiotic alginate beads to
which no microbial inoculum was added. No fluorescence was detected for
all the probes used, except for CY3. The texture of GMA-embedded
alginate sections emitted fluorescence within the filtered wavelength of
CY3 (Figure S3). The observed texture could have resulted from the
tearing of GMA and embedded alginate during cross-sectioning. Such CY3
signals (i.e., false detection of anammox colonies) was successfully
eliminated during data processing with size and circularity filtering
because the autofluorescent textures were large non-circular objects
along the direction of cutting.
The specificity of FISH probes was verified using a pure culture ofNitrosomonas and an anammox enrichment culture. FISH probes
successfully detected AOB and anammox bacteria in both suspended and
encapsulated samples (Figure S4). No NOB colonies were detected in the
granular, non-encapsulated anammox enrichment culture. This is probably
because low concentrations of NOB were present in the culture, which
could have resulted in no observable colonies in the 5
μm2 area under the microscope. Successful detection of
NOB colonies was demonstrated in experiments with encapsulated biomass
by both qPCR and FISH (Figure S5), indicating that NOB must have been
present in the initial culture, just at a low density (see below).
The reproducibility of the cross-section results was examined by
comparing three consecutive 10-µm sections from the same alginate bead.
The sample was collected after 5 days of aerobic incubation of
encapsulated Nitrosomonas . Cross-sections 100 µm apart were
collected for the entire bead. Both the area and count of FISH-detected
AOB colonies increased with depth from the surface from 0 to 300 µm,
followed by a decrease as one moved further toward the center of the
bead (1500 µm), indicating preferred growth of AOB near the surface
(Figure 2 ). One would have expected the microbial counts and
areas to be roughly symmetrical around the core of the bead (e.g., 0 to
1500 µm mirrored in the 1500 to 3000 µm slices). This, however, was not
the case. Indeed, the quality of the sections obtained worsened as the
slices were taken from deeper than the core of the bead. The integrity
of the slices at large depths (>1500 µm) could therefore
not be used for analysis. This was thought to be caused by the fact that
the GMA embedding material was molded into the conical shape of the
microcentrifuge tube (Figure S6). At the tip of the tube, the slices
consisted primarily of the bead itself, but as the cross-sectioning went
deeper into each sample, the slices consisted of more of the GMA
surrounding the bead. It was thought that as a result, greater friction
occurred between the GMA/sample and the blade, compared to the sample
only and the blade, resulting in tearing and poor sample quality. To
ensure the quality of our analysis, we thereafter performed
cross-sectioning only up to the center of each alginate bead sample.
This is an important limitation of this technique, as it is predicated
on the assumption that microbial growth is symmetrical about the core of
a bead. If investigating other embedded geometries or systems where
growth might not be symmetrical (larger beads in a high-rate packed bed
for example), more samples of embedded microorganisms will need to be
analyzed to obtain a clear picture of growth.
When one looks at the 0-1500 µm depths, the largest and densest AOB
colonies were observed around 300 µm from the surface of the alginate
bead. The areas of AOB colonies were statistically compared among the
three neighboring sections, with the three consecutive sections at depthD ± 10 µm treated as technical triplicates representing a single
alginate bead at depth D . No significant differences were
observed for 11 sets of sections (Kruskal-Wallis test, p >
0.05), demonstrating that these three neighboring slices could indeed
act as replicates. Nevertheless, four sets of slices, out of 15, showed
significant differences among the three “replicate” slices (at 300,
400, 1100, and 1200 µm). Part of this appears to be the result of the
larger colony areas and larger variations in colony area observed at
depths 300 and 400 µm where most of the growth occurred.