Addressing Cultivation Barriers in Environmental Microbiomes through Microfluidics and Fluorescent labelling strategies

For over a century, microbial cultivation has contributed significantly to our understanding of microbial physiology, the validation of predicted metabolic functions, and the elucidation of the ecological and biotechnological significance of microorganisms. Nevertheless, only a minuscule fraction of the microbial life present in nature has been successfully cultured in the laboratory. Across all phylogenetic groups, numerous uncultivated bacteria that fail to grow on standard media play critical roles in major biogeochemical processes, such as the cycling of carbon, nitrogen, and other essential elements. Moreover, these microorganisms can biosynthesize novel natural products, including antimicrobial compounds, and influence not only microbial community dynamics, but also the emergence and dissemination of antimicrobial resistance. Culture-independent approaches, such as genome sequencing and metagenomics, have unveiled an astonishing breadth of microbial diversity. However, without cultivated isolates, genome-based predictions remain largely unvalidated and limit our ability to accurately infer gene function and reconstruct metabolic pathways. Even today, the functional characterization of microbial physiology, ecological roles, and bioactive compound production continues to depend on successful cultivation. Recent technological advancements are facilitating efforts to bridge the knowledge gap between the vast microbial diversity and our limited ability to cultivate them in the laboratory. Furthermore, strategies, such as co-culturing unculturable species with helper strains, mimicking natural environmental conditions in vitro by using high-throughput micro cultivation (isolation chip, iChip; droplet microfluidics) and sorting (Fluorescence Activated Droplet Sorting, FADS) platforms have improved access to previously uncultivated taxa.

This PhD thesis addresses cultivation barriers in environmental microbiomes, focusing on marine sediments and soils. By integrating advanced micro-cultivation and fluorescent labelling strategies, we aimed to enable targeted screening and unlock the vast functional potential harboured within uncultured microbial lineages. Application of an in-house iChip led to the successful isolation and characterization of Psychromonas aestuarii sp. nov., a novel bacterial species from estuarine surface sediment. Although antimicrobial activity could not be demonstrated on solid substrates, Psychromonas taxa were also recovered through droplet-based cultivation, and FADS screening after pico-injection with the pathogen Vibrio anguillarum, suggesting its antimicrobial activity at the picoliter scale. Interestingly, this antagonism was not restricted to Psychromonas but was also detected in other uncultivated or poorly characterized genera, and even several unclassified genera. Genomic investigations confirmed the novelty of three species within the genera Vibrio, Enterovibrio, and Sulfitobacter. Interestingly, these new species demonstrated antimicrobial activity against fish or clinically relevant pathogens, pointing that they may produce previously uncharacterized metabolites, however this remain to be elucidated.

An alternative strategy to access biosynthetic potential without relying on cultivation, we further developed Secondary Metabolite Fluorescence in situ Hybridization (SecMet-FISH), a fluorescence-based method for labelling key gene domains, such Adenylation (AD) and Ketosynthase (KS) related to NRPS (Non-Ribosomal Peptide Synthetases) and PKS (Polyketide Synthases) biosynthesis, within complex communities. Coupled with Fluorescence Activated Cell Sorting (FACS), this approach enables targeted detection and extraction for secondary metabolite producers. As a proof of concept, we applied SecMet-FISH to Pseudoalteromonas rubra, detecting the target domains within biosynthetic gene clusters. In addition, we demonstrated its compatibility not only with Gram-negative, but also with Gram-positive bacteria. As SecMet-FISH compromises viability of cells, we further evaluated the applicability of Live-Fluorescence in situ Hybridization (Live-FISH), a transformation-based pre-cultivation labelling technique targeting ribosomes, as a viable alternative. While previously effective in marine environments, its first application to soil microbiomes revealed a taxon-dependent impact on viability. Nevertheless, among 501 surviving taxa, members of Bacillota and the largely uncultivated Planctomycetota retained viability, suggesting their suitability for Live-FISH-guided cultivation.

In conclusion, this research enabled the targeted recovery and functional profiling of previously uncharacterized lineages in environmental microbiomes. This was achieved by fine-tuning microscale cultivation platforms with ultra-high-throughput screening and molecular tools, such as SecMet-FISH and Live-FISH. These approaches not only expand our understanding of microbial ecology and metabolism but also hold significant promise for natural product discovery. In light of the escalating antimicrobial resistance crisis and diminishing returns from traditional pipelines, accessing the biosynthetic potential of uncultivated microbes is no longer a frontier, instead, it is a necessity. Thus, the next generation of cultivation strategies may well drive breakthroughs with significant impact on medicine, ecology, and biotechnology.