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Geerlings et al. Microbiome Res Rep 2024;3:36 https://dx.doi.org/10.20517/mrr.2024.06 Page 11 of 15

compared the use of the food-grade synthetic medium to the mucin medium, which has been used in many
studies to grow A. muciniphila cells for animal studies [18-23] .

The highest growth rate and final optical density were reached in food-grade medium containing the
highest concentration of GlcNAc. In the GlcNAc conditions, the cells were observed to be elongated,
possibly affecting the optical density in these cultivations. Furthermore, PCA analysis showed that in both
the proteome and transcriptome data, the mucin condition clusters separately from the conditions
containing GlcNAc, whereas in the transcriptome data alone, the end-growth phase of condition A also
clusters separately from the other conditions and time points. However, the KEGG metabolism on general
level 1 did not reveal significant differences between fermentor conditions within the different metabolisms.
It may be possible that the regulation of several genes evened out the impact and differences would be
visible at a deeper level. Assessing the transcriptome and proteome data in more detail revealed the
upregulation of proteins and genes involved in stress response in GlcNAc conditions and the pili-associated
system, mucin degradation, and protein sorting systems in mucin conditions.

A shift in propionate to acetate production was observed between the condition containing mucin and the
condition containing glucose and GlcNAc, as well as between the different glucose and GlcNAc conditions.
At the end of the fermentation in mucin, a propionate:acetate ratio of 0.88 was observed. However, with the
decreasing concentration of GlcNAc in the other conditions, the ratio shifted toward more propionate
production. In condition A, the ratio was 1:1, which was similar to previous findings, where a 50:50 ratio of
[25]
glucose and GlcNAc was used as a carbon source . The shift toward a higher propionate:acetate ratio in
conditions B and C is in line with the degradation reactions that were predicted using the genome-scale
[24]
model of A. muciniphila . Therefore, it is important to note that using a lower GlcNAc concentration in
the cultivation of A. muciniphila causes a shift in the propionate:acetate ratio, resulting in an altered short-
chain fatty acid profile.

The carbon recovery values indicated a gap between the carbon sources that were consumed and the energy
and carbon sources that were produced. The carbon recovery values ranged between 70%-73%, excluding
biomass and amino acid formation. Previously, a carbon recovery of 80%-90% has been described for
A. muciniphila cultivated using either GlcNAc, glucose, or N-Acetylgalactosamine (GalNAc) as carbon
[24]
sources . Due to the high amount of pea peptone in this medium, the exact biomass could not be
measured. Therefore, we hypothesize that by including a theoretical portion for biomass, our carbon
recoveries may be in the range of the previously observed carbon recoveries for A. muciniphila. In addition,
considering the elongated cells and the production of exopolysaccharides (EPS), as our transcriptome data
indicate, a portion of the initial carbon concentration available in the medium may be used for cell wall and
EPS production. Next to the transcriptome data, the observed viscosity in the cultures containing GlcNAc
and glucose suggests EPS may be produced in these conditions.

Cell elongation was observed in the fermentations without mucin. As described previously, the cells of
A. muciniphila, when cultivated on mucin medium, are oval-shaped and approximately 0.6 μm in diameter
and 0.7 μm in length . In synthetic medium, the cells were elongated, sometimes 2-3 times
[12]
[Supplementary Figure 1] and compared to mucin, several genes involved in cell division were found to be
upregulated. Three genes, namely cell division inhibitor (Amuc_1176), FtsH (Amuc_0348), and the cell
division trigger factor (Amuc_1052), were significantly upregulated under high and low GlcNAc conditions.
FtsH encodes a metalloprotease, which plays a role in the quality control of integral membrane proteins in
E. coli. In A. muciniphila, this gene may be upregulated due to the elongated membranes that were
observed, increasing the quality control of these membrane proteins. Furthermore, the overproduction of