Periodic Reporting for period 1 - FibRestoration (FibRestoration - Novel specialized probiotics for restoring a healthy fiber-degrading microbiome)
Reporting period: 2023-01-01 to 2025-06-30
Dietary fiber is well recognized as a cornerstone of gut microbiome stability and human health, with established roles in regulating digestion, preventing metabolic disorders, and reducing the risk of chronic diseases. However, the transition toward fiber-rich diets—driven by global trends in conscientious eating and reduced processed food consumption—is often accompanied by digestive discomfort. This is likely due to the scarcity of fiber-degrading bacteria in the gut microbiomes of industrialized populations, particularly those capable of breaking down cellulose, a dominant and recalcitrant component of plant cell walls.
Despite its importance, microbial cellulose degradation in the human gut remains poorly supported. To date, only one known human-associated species, Ruminococcus champanellensis, exhibits crystalline cellulose degradation capabilities, and it is present in less than 3% of individuals in industrialized countries. Consequently, there is an urgent unmet need for microbial solutions that can support the digestion of cellulose and facilitate a smoother dietary transition to fiber-rich nutrition.
Our ERC project seeks to address this gap by discovering, characterizing, and developing human-adapted cellulose-degrading bacteria as next-generation probiotics. Using comparative metagenomics, we identified a rare human-associated genotype closely related to Ruminococcus flavefaciens—a dominant cellulose degrader in the rumen. We demonstrated that this bacterium is more prevalent in rural populations consuming unprocessed diets, suggesting that it is disappearing from industrialized human microbiomes in line with the “disappearing microbiome” hypothesis (Blaser 2017). Functional genomic and biochemical analyses revealed that this human R. flavefaciens-like bacterium harbors potent cellulosomal machinery with advanced fibrolytic capabilities, making it a promising probiotic candidate.
This project offers a novel solution to a widespread dietary-health challenge by expanding the repertoire of beneficial fibrolytic microbes in the human gut. The anticipated impacts include:
1-Health benefit: Facilitating dietary shifts toward high-fiber, whole-food diets by reducing digestive discomfort and enhancing fiber utilization.
2- Scientific advancement: Deepening our understanding of microbial ecology, genome plasticity, and host-microbe adaptation in the gut.
3-Biotechnological innovation: Laying the groundwork for the development of targeted, functional probiotics designed for fiber-rich diets.
4-Societal relevance: Supporting dietary transitions aligned with public health goals and sustainability.
Despite its importance, microbial cellulose degradation in the human gut remains poorly supported. To date, only one known human-associated species, Ruminococcus champanellensis, exhibits crystalline cellulose degradation capabilities, and it is present in less than 3% of individuals in industrialized countries. Consequently, there is an urgent unmet need for microbial solutions that can support the digestion of cellulose and facilitate a smoother dietary transition to fiber-rich nutrition.
Our ERC project seeks to address this gap by discovering, characterizing, and developing human-adapted cellulose-degrading bacteria as next-generation probiotics. Using comparative metagenomics, we identified a rare human-associated genotype closely related to Ruminococcus flavefaciens—a dominant cellulose degrader in the rumen. We demonstrated that this bacterium is more prevalent in rural populations consuming unprocessed diets, suggesting that it is disappearing from industrialized human microbiomes in line with the “disappearing microbiome” hypothesis (Blaser 2017). Functional genomic and biochemical analyses revealed that this human R. flavefaciens-like bacterium harbors potent cellulosomal machinery with advanced fibrolytic capabilities, making it a promising probiotic candidate.
This project offers a novel solution to a widespread dietary-health challenge by expanding the repertoire of beneficial fibrolytic microbes in the human gut. The anticipated impacts include:
1-Health benefit: Facilitating dietary shifts toward high-fiber, whole-food diets by reducing digestive discomfort and enhancing fiber utilization.
2- Scientific advancement: Deepening our understanding of microbial ecology, genome plasticity, and host-microbe adaptation in the gut.
3-Biotechnological innovation: Laying the groundwork for the development of targeted, functional probiotics designed for fiber-rich diets.
4-Societal relevance: Supporting dietary transitions aligned with public health goals and sustainability.
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.
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.
Our work on isolating and cultivating Ruminococcus hominiciens has yielded results that extend the current state of the art in several important ways. First, we have developed high-throughput strategies combining targeted carbon source enrichment, real-time PCR monitoring of species-specific markers, and systematic media optimization, allowing the selective enrichment of a previously uncultivable human gut cellulolytic bacterium. To our knowledge, this represents one of the first successful approaches to stabilize the growth of R. hominiciens ex vivo, providing a foundation for detailed functional and ecological studies.
These results have significant scientific and translational implications. Understanding the fiber-degrading potential of R. hominiciens contributes directly to knowledge of gut microbial ecology, including mechanisms of interspecies interactions and community-derived metabolic dependencies. Furthermore, isolating this bacterium opens new opportunities for applied research, such as the development of probiotics or microbiome-based interventions aimed at enhancing energy harvest and metabolic health in humans. Beyond human gut applications, the strategies developed here are broadly applicable to isolating rare and metabolically specialized microbes from other mammalian hosts, including ruminants, with potential relevance to agriculture and sustainable food systems.
These results have significant scientific and translational implications. Understanding the fiber-degrading potential of R. hominiciens contributes directly to knowledge of gut microbial ecology, including mechanisms of interspecies interactions and community-derived metabolic dependencies. Furthermore, isolating this bacterium opens new opportunities for applied research, such as the development of probiotics or microbiome-based interventions aimed at enhancing energy harvest and metabolic health in humans. Beyond human gut applications, the strategies developed here are broadly applicable to isolating rare and metabolically specialized microbes from other mammalian hosts, including ruminants, with potential relevance to agriculture and sustainable food systems.