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Geerlings et al. Microbiome Res Rep 2024;3:36 https://dx.doi.org/10.20517/mrr.2024.06 Page 7 of 15

Figure 2. Volcano plot comparing the transcriptional response of A. muciniphila in fermentations of the mid-log phase and end-growth
phase of condition A. Only the differentially expressed genes mentioned in the text are labeled here. The complete overview of
differentially expressed genes and their annotation can be found in Supplementary File 1. A. muciniphila: Akkermansia muciniphila.

different growth conditions [Supplementary File 2, Supplementary Figures 5 and 6].

In addition to the transcriptional activity, the catabolism of A. muciniphila in the food-grade medium was
assessed to determine the depletion of the glucose and GlcNAc [Figure 1 and Supplementary Figure 7]. In
all conditions, the main fermentation products included acetate, propionate, and succinate. As shown
previously, the propionate:acetate ratio may differ depending on the cultivation conditions used to grow
[25]
A. muciniphila . In the conditions tested in this study, the ratio of propionate:acetate varied between the
different carbon source ratios used for cultivation [Supplementary Figure 8]. Comparing the final stages of
the bioreactors, the lowest propionate:acetate ratio was found in condition D with a ratio of 0.88, followed
by condition A with a ratio of 1.06, while the ratios in condition B and C were 1.25 and 1.23, respectively,
coinciding with the ratios found in the initial experiments on soy medium. The carbon balances of the
fermentations with glucose and various amounts of GlcNAc were calculated and amounted to
approximately 72% [Supplementary Figure 9].

GlcNAc concentration affects the expression of glycosyltransferases and stress response genes
A limited number of genes were found to be differentially expressed between the mid-log phases of
conditions with different GlcNAc concentrations. Due to the small number of differentially expressed genes
between the mid-log phases of conditions B and C (two significant differentially expressed genes,
Amuc_1139 and Amuc_1140, both of which belong to a glycosyltransferase cluster), here we only compare