Faecalibacterium duncaniae A2-165 growth is strongly promoted by yeast extract and vitamin B5 in cGMP medium

Abstract

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.

FIGURE 1

Experimental set‐up. The study consisted of three parts. The first part investigated the nutritional and production potential of six different yeast‐based nutrients (YBNs) on F. duncaniae A2‐165 in minimal medium (MM) and yeast‐casitone‐fatty‐acids medium (YCFA) containing casein peptone. The growth‐promoting effect of eight different B‐vitamins was tested in the second part and in the third part, F. duncaniae production tests were conducted to investigate the most effective combination of YBN and B‐vitamin in medium formulation. Casein peptone was replaced by yeast peptone in parts two and three to obtain a fully animal‐free medium composition.

FIGURE 2

Impact of six different yeast‐based nutrients (YBNs) in different dosages on carrying capacity (ΔOD) of F. duncaniae A2‐165 in microtiter plates. Bars represent the mean values of max. ΔOD of quadruplicate growth experiments using minimal (A) or yeast‐casitone‐fatty‐acids medium (B) supplemented with low (1.25 g L⁻¹), medium (2.50 g L⁻¹) or high (5.00 g L⁻¹) YBN concentrations or a control without YBN (Ctrl). No growth was detected in minimal medium under YP1 condition (NA). Statistics were performed by unpaired t‐test, comparing for each YBN the ΔOD values of different dosage and medium. Bars marked with an a are significantly different from the low dosage and bars marked with an A from the medium dosage within the same medium and YBN treatment, while bars marked with B are significantly different between the same dosage and YBN treatment of different media.

FIGURE 3

F. duncaniae A2‐165 cell yield, substrate consumption and metabolite production after 28 h batch fermentations in bioreactors using three different yeast extract (YE) types at two dosages. F. duncaniae A2‐165 was cultivated in yeast‐casitone‐fatty‐acids‐like medium containing 20 g L⁻¹ () or 40 g L⁻¹ YE () and 10 g L⁻¹ casein peptone as nitrogen source. Cell yield and metabolite concentrations were measured in the spent media using quantitative real‐time polymerase chain reaction (qPCR) targeting the F. duncaniae A2‐165‐specific region of the 16S rRNA gene and HPLC‐RI, respectively. To obtain the cell number, qPCR values were divided by 6, corresponding to the number of 16S rRNA gene copies per cell. Boxes represent the first quartile, median and third quartile values of the highest measured cell yield (A) and end metabolite concentration (B) of independent triplicated growth experiments. Statistics were performed by two‐way ANOVA following a post‐hoc test using Tukey HST, comparing the means of all YE types, *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 4

Impact of eight B‐vitamins at three different dosages on carrying capacity (ΔOD) and growth rate (ΔOD h⁻¹) of F. duncaniae A2‐165 in microtitre plates. Bars represent the mean values of carrying capacity (ΔOD) (A) and μ_max (B) of quadruplicate growth experiments supplemented with either different B‐vitamins alone or all together (all) at low, medium, or high B‐vitamin concentration and compared to a control without vitamins (Ctrl). Statistics were performed by two‐way ANOVA following a post‐hoc test using Tukey HST, *p < 0.05, **p < 0.01.

FIGURE 5

Growth kinetics of F. duncaniae A2‐165 upon B5 addition to MM medium at different dosages in Hungate tube fermentations. Dots represent the mean values of OD measurements of quadruplicate growth experiments in MM medium supplemented with different concentrations of vitamin B5 (0.4, 1.6, 6.4 and 25.6 mg L⁻¹) and a control lacking B5. Statistics were performed by unpaired t‐test, comparing each concentration with the respective next higher concentration, *p < 0.05 and ***p < 0.001.

FIGURE 6

F. duncaniae A2‐165 cell yield, substrate consumption and metabolite production in Hungate tube fermentations using two different yeast extract (YE) dosages with or without B5 supplementation. Dots and bars represent the mean values of OD measurement (A) and end substrate and metabolite concentrations (B) of triplicate growth experiments in Hungate tubes containing MM supplemented with low (1.25 g L⁻¹) and high YE2 doses (5.00 g L⁻¹) and combined with and without vitamin B5 addition (6.4 mg L⁻¹). Statistics were performed by unpaired t‐test, comparing for each YE concentration the OD values and metabolite concentrations with and without B5 addition, *p < 0.05, **p < 0.01 and ***p < 0.001.

FIGURE 7

F. duncaniae A2‐165 cell yield, substrate consumption and metabolite production were enhanced under B5 supplementation in controlled bioreactor experiments. F. duncaniae A2‐165 was cultivated in controlled batch bioreactors in MM medium containing 40 g L⁻¹ YE2, 10 g L⁻¹ YP2 and 51.2 mg L⁻¹ vitamin B5 and compared to a control without B5 addition. Cell yield (A) and metabolite concentrations (B) were measured in the spent media of each bioreactor over 30 h batch fermentation using quantitative real‐time polymerase chain reaction targeting the F. duncaniae A2‐165‐specific region of the 16S rRNA gene and HPLC‐RI, respectively. The values were normalized by six 16S rRNA gene copies to obtain the cell number per mL spent media. Dots represent average cell concentration at different time points of triplicated growth experiments, except for time point 18, 20 and 22 h that were only assessed for one replicate (A). Bars represent average values of end metabolite concentration (B) of triplicate growth experiments. Statistics were performed by unpaired t‐test, comparing the B5 group to controls, p < 0.01, *p < 0.05 and **p < 0.01.