An optimized isolation chip approach targeting recovery of soil spore-forming bacteria

Spore-forming bacteria are key candidates for applications in biotechnology and biocontrol due to their resistance to physical and chemical stress and metabolic diversity. However, their recovery from soil is often hindered by limitations in conventional cultivation methods. The isolation chip (iChip) has expanded access to previously uncultivable microorganisms. This study aimed to optimize pretreatment and incubation conditions for the selective isolation of spore-forming bacteria using a modified iChip protocol. Soil samples were collected from three environments in Brittany, France, during two seasons. Pretreatment included sequential washing, sonication, centrifugation, and heat treatment at 80 °C to select spores while minimising soil particules to allow accurate microscopic quantification. Stainless steel iChip plates with 96 wells were designed to improve membrane sealing and handling. Plates were inoculated with spore-enriched suspensions and incubated in situ for up to six weeks before recovery at the laboratory at two temperatures (15 °C and 30 °C).A total of 757 isolates were obtained, with 52% identified as spore-formers, a substantial increase compared with unmodified iChip workflows (∼21%). Taxonomic analysis revealed 79 genera, including Bacillus, Paenibacillus. Phylogenetic analysis highlighted 51 isolates with less than 98.65% 16S rRNA similarity to known species, suggesting potential novelty. The recovery of spore-formers can be achieved through selective pretreatments. Even rare taxa can be revealed with this in situ cultivation. The optimized method offers a promising framework for the recovery of uncultured spore-forming bacteria with potential applications in biocontrol, biotechnology, and natural product discovery.