Project description:Several gut microbial species within the Faecalibacterium genus have emerged as promising next-generation probiotics (NGP) due to their multifunctional protective effects against gastrointestinal and systemic disorders. To enable clinical studies and further applications, improved methods for cultivating Faecalibacterium must be developed in compliance with current Good Manufacturing Practice regulations, which is complicated by its oxygen sensitivity and complex nutritional requirements. Different yeast-based nutrients (YBNs), including yeast extracts (YEs) and yeast peptones (YPs), are ubiquitously used when cultivating microbes to supply a broad range of macro- and micronutrients. In this study, we evaluated six experimental YBNs, namely three YEs, two YPs and a yeast cell wall product (YCW), and eight B-vitamins in the cultivation of Faecalibacterium duncaniae A2-165, former Faecalibacterium prausnitzii, using growth assays in microtitre plates, dose-effect studies in Hungate tube fermentations and fully controlled bioreactor experiments. We demonstrated that YEs promote F. duncaniae A2-165 growth in a nutritionally limited medium, while YPs and YCW lacked essential growth factors for enabling cell propagation. High cell density was obtained in controlled bioreactors using a medium containing 2-4% of a selected YE and 1% casein peptone (3.4 ± 1.7 × 109 -5.1 ± 1.3 × 109 cells mL-1 ). Among all tested B-vitamins, we identified B5 as a strong growth promoter. Replacing casein peptone with YP and supplementing with vitamin B5 further increased biomass by approximately 50% (6.8 ± 1.7 × 109 cells mL-1 ). Hence, empirical selection of YE, YP and B5 allowed formulation of a high-yielding animal allergen-free nutritive medium to produce F. duncaniae A2-165. Selecting nutritionally suitable YBNs and combining these with other key nutrients are important steps for optimizing production of NGP with high yields and lower cost.
Project description:The promising next-generation probiotic Faecalibacterium prausnitzii is one of the most abundant acetate-consuming, butyrate-producing bacteria in the healthy human gut. Yet, little is known about how acetate availability affects this bacterium's gene expression strategies. Here, we investigated the effect of acetate on temporal changes in the transcriptome of F. duncaniae A2-165 cultures using RNA sequencing. We compared gene expression patterns between two growth phases (early stationary vs. late exponential) and two acetate levels (low: 3 mM vs. high: 23 mM). Only in low-acetate conditions, a general stress response was activated. In high-acetate conditions, there was greater expression of genes related to butyrate synthesis and to the importation of B vitamins and iron. Specifically, expression was strongly activated in the case of the feoAABC operon, which encodes a FeoB ferrous iron transporter, but not in the case of the feoAB gene, which encodes a second putative FeoAB transporter. Moreover, excess ferrous iron repressed feoB expression but not feoAB. Lastly, FeoB but not FeoAB peptides from strain A2-165 were found in abundance in a healthy human fecal metaproteome. In conclusion, we characterized two early-stationary transcriptomes based on acetate consumption and this work highlights the regulation of feoB expression in F. duncaniae A2-165.