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.