High-throughput cultivation and isolation of environmental anaerobes using selectively permeable hydrogel capsules

Abstract

Over the past two decades, metagenomics has greatly expanded our understanding of microbial phylogenetic and metabolic diversity. However, most microbial taxa remain uncultured, hindering research and biotechnological applications. Isolating environmental anaerobes using traditional methods is particularly cumbersome and low throughput. Here, we present a novel, high-throughput approach for the cultivation and isolation of anaerobes, which involves trapping and growing single microbes within selectively permeable hydrogel capsules followed by fluorescence-activated cell sorting to distribute compartmentalized isolates into liquid medium for further growth. We show that diverse anaerobes can grow within capsules and that slower-growing ones (e.g. methanogens) can be enriched with this platform. We also applied this approach to isolate anaerobes from soil, including strains of the sulfate-reducing bacteria Desulfovibrio desulfuricans and Nitratidesulfovibrio vulgaris. Overall, this work introduces a robust, high-throughput alternative to traditional techniques for isolating environmental anaerobes and expands the emerging set of microfluidics-based tools for the cultivation of novel taxa.

Keywords: acetogenic bacteria; anaerobic microorganisms; hydrogel capsules; isolation; methanogens; microfluidics; soil microbiome; sulfate-reducing bacteria.

Figure 1

Single-cell encapsulation and growth of anaerobic microorganisms within capsules. (A) Overview of the capsule generation process (inset: microscope image of microfluidic chip during capsule generation). (B) Schematic and microscopy image showing a capsule enclosing a single bacterium (Nitratidesulfovibrio vulgaris). (C) Microscopy images of anaerobic strains growing within capsules. From left to right: Escherichia coli TB205 (18 h incubation), Paraclostridium bifermentans EML (24 h incubation), Shewanella oneidensis MR-1 (24 h incubation), Nitratidesulfovibrio vulgaris (2-day incubation), Thermoanaerobacter kivui (24 h incubation), Methanosarcina acetivorans C2A (18-day incubation) and Methanococcus maripaludis MM901 (5-day incubation). The type of anaerobic metabolism used by the strains is specified below each image. CP: Core polymer, SP: Shell polymer.

Figure 2

Comparison of different platforms to cultivate soil microbial taxa. (A) Schematic overview of the experiment. Microorganisms are extracted from a paddy soil (A_BH) and grown anaerobically for 1 week in different cultivation platforms. After cultivation, microbial cells are harvested and all communities (initial and 1-week cultures) are profiled with metagenomics. (B) Richness of microbial communities. Bar lengths indicate mean richness values (number of MAGs) and error bars show standard deviation (N = 3 replicates). Individual replicate values are shown as dots. Different letters indicate significant difference at p < 0.05 between treatments (Tukey’s test). (C) Nonmetric multidimensional scaling (NMDS) plot illustrating differences across microbial communities, based on Bray–Curtis dissimilarity values derived from relative abundance data. The reads that did not map to any MAGs were removed from the analysis and relative abundance values were rescaled accordingly. Each MAG is shown as a triangle, whereas communities are depicted as colour dots. Different colours indicate different cultivation conditions (three replicates per condition). The stress value of the ordination was 0.0605. (D) UpSet plot showing taxonomic similarities across the different communities. The set size indicates, for each condition, the total number of MAGs who are present in at least one replicate. Intersection size shows the number of MAGs shared across the samples indicated with dots. (E) Circular maximum likelihood phylogenetic tree illustrating the diversity of microbial taxa (MAGs) present in the communities, based on the relative abundance values (average of 3 replicates). The colour of the MAG name indicates its taxonomy at the phylum level and its presence/absence in the different communities is shown in the outer circles (one specific colour is attributed to each sample type). Two abundance ranges (<5% and > 5%) are specified with different colour shades. Symbols indicate MAGs with specific metabolic capabilities (acetogens, methanogens, aerobes and sulfate-reducers). The scale represents the branch length. SRB: Sulfate-reducing bacteria.

Figure 3

Dynamics of soil-derived microbial community within capsules over cultivation time. (A) Taxonomy barplot showing changes in the microbial community extracted from soil (A_BH) throughout cultivation in capsules in MSM10 medium. The MAGs are shown at the class level (see colour code in legend). Taxa labelled as “unclassified bacteria” are MAGs that were not assigned to a known phylum. (B) Taxonomy barplot showing changes in the abundance of facultative anaerobic taxa throughout cultivation in capsules. The taxonomy (family, genus and species) of each MAG is indicated, see colour code in legend.

Figure 4

Growth of strictly anaerobic taxa within capsules in enrichment medium. Soil (A_BH) microorganisms were individually entrapped in capsules and cultivated at 30°C in pre-reduced enrichment media (either DSMZ 135 or DSMZ 311 medium) in serum bottles filled with 80% H₂, 20% CO₂. After 7 and 13 days, an aliquot of the culture was sacrificed for taxonomic profiling (mOTUs). Barplots show the enrichment of (A) Acetoanerobium sticklandii, (B) Terrisporobacter glycolicus and (C) Methanosarcina barkeri within capsules, between 7 and 13 days of incubation. The data in (A) and (B) are from the same culture.