Figure 4. Quantities of the 16S rRNA gene and 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<0.05).
Similar to this study, research on encapsulated 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 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 ofN. 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 (Figure 5 ). AOB
colonies were smaller and evenly distributed across the bead depth
(Figure 5a & 5b ), whereas anammox bacteria colonies were
larger in size and exhibited large variations in colony count among
different beads sampled (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 μm 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 (Figure 5c & 5d ). For NOB, a slight increase in colony
size and count was also observed from 0-700 μm 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
μm in depth (Figure 5g &
5h ). Similar to the experimental results with only encapsulatedN. europaea , qPCR did not provide information on the changes in
the spatial distribution of encapsulated colonies (Figure 4b ).
No significant change was detected in the 16S rRNA gene copy numbers
before and after the incubation; however, the copy numbers foramoA , hzsA , and nxrB all decreased significantly
(p<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 μm) 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 μm to be augmented by data from
1500-3000 μm. 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.