Difficult-to-culture micro-organisms specifically isolated using the liquid-liquid co-culture method - towards the identification of bacterial species and metabolites supporting their growth.

In this study, the liquid-liquid co-culture method was applied using faecal samples and specific bacterial species as growth-supporting bacteria. We aimed to isolate new, difficult-to-culture bacterial species using metabolites produced by supportive bacteria to promote the growth of small bacteria selected using filter treatment. This study aimed to identify the supporting bacteria and their metabolites that promote the growth of these isolates. Analysis of the 16S rRNA gene sequences of the isolates obtained by co-culture revealed that they were spp., spp. and . spp. and spp. were isolated from several faecal samples, suggesting that they were specifically isolated using this culture method. We focused on spp. isolated from several faecal samples with unique shapes, from long to short or thin cells. The growth of spp. was not promoted by co-culture on the agar medium, suggesting that growth was only promoted by liquid-liquid co-culture. The growth of the selected small-sized spp. was promoted by co-culture, whereas the growth of the unfiltered long-cell sp. strain was suppressed by co-culture. The selected small spp. did not grow when the supporting bacterial supernatant was added, suggesting that the supporting bacteria and spp. had a symbiotic relationship through the continuous exchange of metabolites. Co-cultured supporting bacteria (diluted faecal samples) with selected small-sized spp. were predominantly and , compared with monoculture diluted faecal samples. We further confirmed the growth of filtered spp. by co-culturing them with and . Additionally, when and were co-cultured with the selected small spp., some nutrients and metabolites were reduced. Decreased metabolites were added to the medium, and selected small-sized spp. were cultured, but spp. did not grow. Therefore, it was again strongly suggested that continuous co-culturing with the supporting bacteria was important for the growth of spp. The liquid-liquid co-culture method used in this study can be used to isolate new and unique bacterial species from any environment, not just the gut microbiome. Furthermore, this co-culture method helped identify supporting bacteria and understand metabolite variations.