Within the scope of isolating and cultivating novel ruminococcal fiber-degrading microbes—key players in the rumen microbiome and other mammalian hosts—particular attention was given to Ruminococcus hominiciens, a recently identified cellulolytic, cellulosome-producing bacterium of the human gut (Morais et al., 2024). R. hominiciens is rare in industrialized populations but remains prevalent in rural and hunter-gatherer communities. Its persistence and activity are of interest, as it may contribute to host energy harvest. To advance understanding of its ecological role and fiber-degrading potential, we sought to isolate and cultivate R. hominiciens from human fecal samples.
Guided by prior knowledge of the bacterium’s functional potential, supported by biochemical assays, we tested its ability to degrade and attach to cellulose and break down corn arabinoxylan. Although revival was initially successful, it consistently failed to survive successive transfers, suggesting dependence on a competitor or a missing community-derived factor—potentially a metabolite produced by other microbes during early growth.
We implemented a wide range of cultivation strategies, including manual and robotic isolation workflows, growth on multiple selective and non-selective media, extended incubation under varying atmospheric conditions, supplementation with diverse carbon sources, vitamins, and cofactors, and treatment with antibiotics to reduce competitors. Despite these efforts, the bacterium could not be maintained in pure culture, reinforcing its reliance on community interactions.
We then focused on identifying carbon sources that promoted enrichment. Using M2 medium as the basal formulation, individual carbon sources were tested at 0.2% and 1% (w/v) with human fecal inocula. Monitoring R. hominiciens via ScaC gene copy number, we observed strong enrichment with soybean flour after two transfers. Enriched cultures were sampled for 16S rRNA sequencing, and bacterial stocks were prepared.
To systematically identify optimal growth conditions, we adopted a high-throughput approach using Biolog™ PreBioM and Anaerobic MediaMatcher plates, testing a broad range of carbon sources and media formulations. Fecal glycerol stocks were pre-enriched in M2 medium with cellulose, then inoculated into screening plates under anaerobic conditions. Real-time PCR quantification of ScaC gene copy number was performed over five transfers, identifying nine carbon sources that supported improved growth.
Subsequent optimization involved varying dilution levels and transfer intervals in M2 + 1% soybean flour medium. Cultures inoculated at 1×, 5×, and 10× dilutions and transferred every 1–3 days were monitored daily by real-time PCR. After two transfers, mean Cp values decreased from 30 to 20 (~1000-fold enrichment), and after nine transfers, cultures stabilized at Cp < 25.
Two enriched cultures (mean Cp < 20) were further diluted and plated on M2 + 1% soybean flour agar. Ninety-six colonies from each plate were transferred to Omni plates and are currently undergoing screening by real-time PCR and 16S rRNA sequencing. These results give strong confidence that the target bacterium will be successfully isolated soon, marking a major step toward understanding R. hominiciens biology and its role in fiber degradation.
