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\begin{document}

\title{Quantification of depth-dependent microbial growth in encapsulated
systems}



\author[1]{Zhiyue Wang}%
\author[2]{Satoshi Ishii}%
\author[3]{Paige Novak}%
\affil[1]{University of Hawai'i at Manoa Department of Civil and Environmental Engineering}%
\affil[2]{University of Minnesota Twin Cities Department of Soil Water and Climate}%
\affil[3]{University of Minnesota Twin Cities Department of Civil Environmental and Geo-Engineering}%


\vspace{-1em}



  
  \date{May 26, 2024}


\begingroup
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\selectlanguage{english}
\begin{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 \emph{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 ( \emph{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.%
\end{abstract}%



\sloppy


\textbf{Quantification of depth-dependent microbial growth in
encapsulated systems}

Zhiyue Wang\textsuperscript{a, b *}, Satoshi Ishii\textsuperscript{c},
Paige J. Novak\textsuperscript{d}

a. Department of Civil and Environmental Engineering, University of
Hawai'i, Honolulu, Hawai'i, USA

b. Water Resources Research Center, University of Hawai'i, Honolulu,
Hawai'i, USA

c. Department of Soil, Water and Climate, University of Minnesota, St.
Paul, Minnesota, USA

d. Department of Civil, Environmental, and Geo-Engineering, University
of Minnesota, Minneapolis, Minnesota, USA

* Corresponding authors. Address: Holmes Hall 346, 2540 Dole Street,
Honolulu, HI 96822, USA. Email: zhiyue@hawaii.edu

\textbf{Keywords}

Encapsulation, fluorescence in situ hybridization, cross-section,
enumeration, colony, \emph{Nitrosomonas europaea} , anammox



\textbf{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 \emph{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 (\emph{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.

\textbf{Introduction}

Microbial encapsulation is a versatile environmental biotechnology with
applications ranging from nutrient removal and resource recovery from
wastewater to bioremediation of contaminants . The permeable matrices
used as encapsulants not only selectively retain microorganisms, but
also protect them against potential inhibitors in the external
environment . Nevertheless, encapsulants create physical barriers to
directly access microbes for analysis. Existing tools have diverse
capabilities in microbial and morphological analysis but are limited
when applied to elucidate the growth of multiple encapsulated
microorganisms across a temporal and spatial span.

Due to the similarity between biofilm and encapsulated systems,
microscopic methods are widely used in both systems to reveal the
morphology and assembly of microbes. For instance, optical microscopy,
optical coherence tomography, and electron microscopy have been used to
reveal structures in biofilm systems , which are applicable to
encapsulated systems as well. Optical and fluorescent dyes can be used
to enhance the contrast between the encapsulant and encapsulated
microorganisms . Live/dead staining is also available to identify living
encapsulated cells . Meanwhile, encapsulants are inherently different
from extracellular polymeric substances in biofilms. It is challenging
to preserve their structure during the pretreatment of samples. To
prepare samples for microscopy, fixation and dehydration procedures can
cause shrinkage and even destruction of hydrogel-derived encapsulants. .
Electron microscopy can better preserve encapsulant structures and
improve the resolution of submicron morphological details; it cannot,
however, differentiate between microbial species.

In addition to microscopic methods, molecular and physio-chemical
techniques are used to quantify the growth of encapsulated
microorganisms. Measurable parameters include culturable colony forming
units (CFU), DNA copies, cell protein content, and dry cell weight .
Sequencing and quantitative amplification (e.g., quantitative polymerase
chain reaction (qPCR)) of genetic material can further identify and
quantify the targeted components of an encapsulated microbial community
. Nevertheless, extraction of cell contents or DNA from encapsulated
microorganisms often requires homogenization and removal of the
encapsulant. As a result, the spatial information of cell distribution
within the encapsulant is lost during such a process. Modifications of
the procedure with selective shearing of surface layers and sorting of
size fractions have been demonstrated with granular activated sludge;
however, these modifications are difficult to apply on encapsulated
systems, as encapsulants are much harder to selectively disassemble.

The main objective of this study was to develop a novel method for
measuring spatial and temporal changes in the encapsulated growth of
various microorganisms. Such a method can potentially provide
information to validate predictions from mathematical models, allowing
more predictable applications of microbial encapsulation technologies in
environmental engineering .

\textbf{Methods and Materials}

2.1 Microbial encapsulation and batch test setup

A pure culture of \emph{Nitrosomonas europaea} ATCC
19718\textsuperscript{T} and an enrichment culture of anammox bacteria
were used as inocula for microbial encapsulation. The enrichment culture
was granular in nature and collected from a bench-scale sequencing batch
reactor under steady-state operation . An equal volume of 4\% sodium
alginate solution and the inoculum culture was completely mixed before
crosslinking in a 4\% calcium chloride solution to form spherical beads
as described previously (Wang et al., 2022). The inocula were about 20
and 500 mgTSS/L for the \emph{Nitrosomonas} culture and anammox
enrichment respectively. Crosslinked alginate beads had diameters of 2.4
-- 2.8 mm.

Batch bioreactors with encapsulated biomass were established as
previously reported . For tests with encapsulated \emph{Nitrosomonas} ,
ammonia (50 -- 150 mgN/L) was added daily to the synthetic culture
medium as NH\textsubscript{4}Cl, whereas an equal amount of ammonia (as
NH\textsubscript{4}Cl) and nitrite as NaNO\textsubscript{2} (50 -- 100
mgN/L each) was added to the medium for tests with the anammox
enrichment culture. Triplicate batch reactors (200 mL) were placed on an
orbital shaker at 100 rpm and operated for a week. Sponge caps were used
to allow diffusion of air into the reactors, and dissolved oxygen
concentrations were kept between 0.5 to 4.0 mg/L. Water samples (1.5 mL)
were collected daily, filtered, and analyzed with spectrophotometric
methods for ammonia, nitrite, and nitrate . Six beads were sampled (3
for imaging and 3 for DNA extraction) and processed immediately before
and after each batch incubation.

2.2 Sample preparation, cross-sectioning, and FISH procedure

Alginate bead samples were fixed immediately after collection with 3\%
(w/v) paraformaldehyde. Each alginate bead was placed in the conical
bottom of a 1.5-mL microcentrifuge tube and infiltrated stepwise with
25, 50, 75, and 100\% glycolmethacrylate (GMA) as previously described .
GMA-infused samples were thus molded into the tip of the microcentrifuge
tube. Following the solidification of GMA, the bottom of the
microcentrifuge tubes was cut open and placed on a microtome (Microm
HM505E). Cross-section cutting (10-\selectlanguage{greek}µ\selectlanguage{english}m thickness) was conducted starting
from the surface of each bead. Three consecutive sections were collected
upon every 100-\selectlanguage{greek}µ\selectlanguage{english}m advancement until the entire bead was completely
cross-sectioned (\textbf{Figure 1} ). The collected sections were placed
on multi-well slides pre-coated with a 0.1\% poly-L-lysine solution.
Fluorescence \emph{in situ} hybridization (FISH) was conducted with
oligonucleotide probes targeting ammonia oxidizing bacteria (AOB),
nitrite oxidizing bacteria (NOB), and anammox bacteria (\textbf{Table 1}
). The hybridization was conducted with 35\% formamide at 46 \selectlanguage{ngerman}°C for 2
hours. An antifade mountant with DAPI (ProLong\selectlanguage{english} Gold, Invitrogen) was
used as a DNA counter stain. Alginate beads without microbial inoculum
were analyzed by the cross-section FISH method as negative controls
using each fluorescence probe to check for autofluorescence. The anammox
enrichment culture was used as a positive control for each fluorescence
probe, as it was assumed to contain AOB, NOB, and anammox bacteria.
Prior to fixation, the granular anammox culture was homogenized for one
minute with a handheld homogenizer to break apart large granules. The
anammox biomass was then fixed and used for FISH as described above,
without the embedding material present. GMA has been previously shown to
not interfere with, but rather, to improve the sensitivity of FISH
analysis .\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1.} Overview of the cross-section and FISH procedures.

\textbf{Table 1.} Oligonucleotide probes used for FISH. (Adopted from
Shi et al., 2016)\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\textbf{Target Group} & \textbf{Probe} & \textbf{Dye} & \textbf{Sequence
5'3'}\tabularnewline
\midrule
\endhead
NOB & Ntspa662 & Cy5 & GGA ATT CCG CGC TCC TCT\tabularnewline
& Ntspa712 & Cy5 & CGC CTT CGC CAC CGG CCT TCC\tabularnewline
AOB & Nso1225 & FAM & CGC CAT TGT ATT ACG TGT GA\tabularnewline
& NEU & FAM & CCC CTC TGC TGC ACT CTA\tabularnewline
& Cluster6a 192 & FAM & CTT TCG ATC CCC TAC TTT CC\tabularnewline
Anammox bacteria & Amx820 & Cy3 & AAA ACC CCT CTA CTT AGT GCC
C\tabularnewline
\bottomrule
\end{longtable}

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
(\textgreater{}2x2 pixels) and circularity (\textgreater{}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 mm\textsuperscript{2}) and
the thickness of the cross-section (10 \selectlanguage{greek}μ\selectlanguage{english}m). 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 \selectlanguage{ngerman}°C to obtain cell pellets, which were washed twice
and resuspended in buffer solution (10\selectlanguage{english} mM Tris, 0.1\selectlanguage{english} mM EDTA) for storage
at --20\selectlanguage{english} degC. 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 \textgreater{}90\%,
except for the 16S rRNA gene (\textgreater{}85\%).

\textbf{Results and Discussion}

Method validation with encapsulated \emph{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
of\emph{Nitrosomonas} 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
\selectlanguage{greek}μ\selectlanguage{english}m\textsuperscript{2} 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-\selectlanguage{greek}µ\selectlanguage{english}m sections from the same alginate bead.
The sample was collected after 5 days of aerobic incubation of
encapsulated \emph{Nitrosomonas} . Cross-sections 100 \selectlanguage{greek}µ\selectlanguage{english}m 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 \selectlanguage{greek}µ\selectlanguage{english}m,
followed by a decrease as one moved further toward the center of the
bead (1500 \selectlanguage{greek}µ\selectlanguage{english}m), indicating preferred growth of AOB near the surface
(\textbf{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 \selectlanguage{greek}µ\selectlanguage{english}m mirrored in the 1500 to 3000 \selectlanguage{greek}µ\selectlanguage{english}m 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 (\textgreater{}1500 \selectlanguage{greek}µ\selectlanguage{english}m) 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 \selectlanguage{greek}µ\selectlanguage{english}m depths, the largest and densest AOB
colonies were observed around 300 \selectlanguage{greek}µ\selectlanguage{english}m 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
depth\emph{D} \selectlanguage{ngerman}± 10 \selectlanguage{greek}µ\selectlanguage{english}m treated as technical triplicates representing a
single alginate bead at depth \emph{D} . No significant differences were
observed for 11 sets of sections (Kruskal-Wallis test, p \textgreater{}
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 \selectlanguage{greek}µ\selectlanguage{english}m). 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 \selectlanguage{greek}µ\selectlanguage{english}m where most of the growth occurred.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{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 \textless{}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 \selectlanguage{greek}µ\selectlanguage{english}m equaling the center/core of the bead.

Encapsulated growth of \emph{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
encapsulated\emph{Nitrosomonas} consumed ammonium at 42 \selectlanguage{ngerman}± 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 \selectlanguage{greek}µ\selectlanguage{english}m (bead core) with an average colony size of 20
\selectlanguage{greek}µ\selectlanguage{english}m\textsuperscript{2} and an average density of 1,360 colonies
mm\textsuperscript{-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\textgreater{}0.05). This is also expected, because the doubling time
of encapsulated \emph{Nitrosomonas}is 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
encapsulated\emph{Nitrosomonas} for 5 days. Three different beads were
collected and cross-sectioned at the end of the incubation. Three
consecutive slices (at depth \emph{D} \selectlanguage{ngerman}± 10 \selectlanguage{greek}µ\selectlanguage{english}m, where \emph{D} is every
100 \selectlanguage{greek}µ\selectlanguage{english}m until 1200 \selectlanguage{greek}µ\selectlanguage{english}m) from the same bead were analyzed and used as
replicates to represent the colony profile at each depth \emph{D} . As
observed previously, the initial AOB numbers and colony sizes were
evenly distributed through the bead depth of 0 to 1200 \selectlanguage{greek}µ\selectlanguage{english}m (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 (\textbf{Figure 3} ). Preferential
growth of encapsulated colonies was detected near the surface (300-500
\selectlanguage{greek}µ\selectlanguage{english}m), 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 \textless{} 0.05), however, the
colony counts were not significantly different among the three beads
(Kruskal-Wallis test, p \textgreater{} 0.05).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{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 (\textbf{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 \emph{amoA} copy numbers after the 1-day incubation
(p\textless{}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\textless{}0.05, student t-test). Increase in the copy
number \emph{amoA} again agrees with the cross-section FISH results
showing the growth of AOB colonies near the bead surface (\textbf{Figure
3} ). The difference between increases in the 16S rRNA gene
and\emph{amoA} 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).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4.} Quantities of the 16S rRNA gene and \emph{amoA} in
the beads collected from (a) encapsulated AOB and (b) encapsulated
anammox batch reactors before and after incubation. Error bars indicate
the variations between triplicate alginate beads collected at each time
point. Asterisks indicate statistical differences by pairwise student
t-tests (p\textless{}0.05).

Similar to this study, research on encapsulated \emph{Nitrobacter} in
carrageenan showed that the maximum biomass concentration and colony
size were found near the encapsulant surface . These phenomena can be
explained and modeled by oxygen and ammonium diffusion limitation . In
addition, in the research described herein, the diffusion of calcium
cations into the alginate matrix was used to crosslink the encapsulant,
which can produce a harder layer near the surface ; This could explain
the lack of obvious colony eruption observed by others that was not
observed herein, perhaps as a result of a harder outer layer of
alginate, which may have provided resistance for encapsulated colonies
to erupt out of the encapsulant, or even perhaps to grow.

Extended application with encapsulated Anammox enrichment

Our new method was applied to analyze anammox bacteria co-encapsulated
with \emph{N. europaea} to demonstrate its ability to simultaneously
track the growth of multiple microorganisms. The inoculum consisted of a
granular anammox enrichment culture and the pure suspended culture
of\emph{N. europaea} . The difference in the original biomass forms
(granular sludge vs. suspended cells) most likely resulted in distinct
characteristics in colony size and count observed between encapsulated
AOB and anammox bacteria before incubation (\textbf{Figure 5} ). AOB
colonies were smaller and evenly distributed across the bead depth
(\textbf{Figure 5a \& 5b} ), whereas anammox bacteria colonies were
larger in size and exhibited large variations in colony count among
different beads sampled (\textbf{Figure 5e \& 5f} ).

After incubation, nitrite and ammonium were both consumed after each
addition (Figure S1). Encapsulated AOB increased in colony counts,
particularly in the samples analyzed from 0 to 700 \selectlanguage{greek}μ\selectlanguage{english}m in depth. This is
consistent with what was observed with the AOB-only encapsulants, with
growth near the surface of the bead. One of the sampled beads (sample C)
deviated significantly from the other two beads in colony counts across
depth (\textbf{Figure 5c \& 5d} ). For NOB, a slight increase in colony
size and count was also observed from 0-700 \selectlanguage{greek}μ\selectlanguage{english}m in depth after incubation
(Figure S5). Meanwhile, the colony size of anammox bacteria decreased
slightly after the 5-day incubation period. Less variation in the colony
count of anammox bacteria was observed among sampled beads after the
incubation, with the largest number of colonies seen around 300-500 \selectlanguage{greek}μ\selectlanguage{english}m
in depth (\textbf{Figure 5g \& 5h} ). Similar to the experimental
results with only encapsulated\emph{N. europaea} , qPCR did not provide
information on the changes in the spatial distribution of encapsulated
colonies (\textbf{Figure 4b} ). No significant change was detected in
the 16S rRNA gene copy numbers before and after the incubation; however,
the copy numbers for\emph{amoA} , \emph{hzsA} , and \emph{nxrB} all
decreased significantly (p\textless{}0.05, student t-test), indicating a
decrease in average concentrations of AOB, NOB, and anammox bacteria.
One sees much more nuance in the cross-sectioning and FISH results,
where some beads showed growth, including growth at the bead surface,
and one bead (B) showed an overall decrease in colony counts after
incubation.

Similar to this study, reported the stratification of anammox bacteria
and comammox bacteria in a polyvinyl alcohol-alginate matrix. The
co-encapsulation system was also used as an ``engineered biofilm'' to
study the ecological cooperation between a pure culture and an anammox
enrichment. Spatial segregation of comammox in the outer layer and
anammox in the inner layer was driven by oxygen and nutrient
concentration gradients across depth. FISH analysis was performed to
qualitatively determine the growth of encapsulated microbes near the
surface . Our method provides quantitative information about the size
and number of encapsulated colonies at a given time and depth, which
reflects the growth of microbial populations. The depth at which
microbes grow within the encapsulant can be influenced by a combination
of factors, including microbial growth kinetics, mass transfer of
substrates, and competition and/or synergistic relationships between
different groups of microorganisms. Mathematical models are useful to
simulate the combined effect of such complex interactions ;
nevertheless, the predictions from modeling need to be experimentally
validated. The method developed in this study should be extremely useful
for this purpose. In conjunction with other analytical tools, such as
microsensors that provide the concentration gradients of substrates ,
our method should be able to advance our understanding of microbial
ecology in encapsulated systems. Due to the resemblance between biofilms
and encapsulated systems, our method is also potentially applicable to
biofilm systems for quantifying colonies across depth. Such advances
could greatly enhance our understanding of microbial colony formation
across spatial and temporal scales, expanding predictable environmental
engineering applications of both encapsulated and biofilm systems in
contaminant remediation , resource recovery , and cell preservation and
transportation .

While the cross-section method developed in this study shows promise as
a tool to quantify encapsulated growth of microbial colonies, there are
still some limitations to the method and potential improvements needed.
For instance, contamination during slicing is difficult to avoid,
resulting in the carryover of cells to deeper cross-sections .
Additionally, the low image quality of the cross-sections deeper in the
beads (1500 to 3000 \selectlanguage{greek}μ\selectlanguage{english}m) may be solved by improvements in the durability
of the embedding materials, molding techniques, and microtome slicing
procedure. This, for spherical beads, would essentially serve as another
replicate, increasing the power of one's observations in a symmetrical
system by enabling data from 0-1500 \selectlanguage{greek}μ\selectlanguage{english}m to be augmented by data from
1500-3000 \selectlanguage{greek}μ\selectlanguage{english}m. Confocal fluorescence microscopy may also be used to
improve the resolution of cross-section FISH images. Future work could
focus on improving the method to allow for a more comprehensive analysis
of the growth and behavior of microorganisms within encapsulant
matrices.\selectlanguage{english}
\begin{figure}[H]
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\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

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\textbf{Figure 5.} The distribution of size and count of AOB colonies
across depth in three different alginate beads before (a, b) and after
(c, d) the 5-day incubation. The distribution of anammox bacteria
colonies in each corresponding bead before (e, f) and after (g, h) the
5-day incubation.

\textbf{Acknowledgement}

This work was supported by the Biocatalysis Initiative of the University
of Minnesota. We would like to express our gratitude to Dr. Jake Bailey
in the Department of Earth and Environmental Sciences at the University
of Minnesota for generously granting us access to his fluorescence
microscope and laboratory facilities, which facilitated and enriched the
execution of this study.

\textbf{Reference}\selectlanguage{english}
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