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Geerlings et al. Microbiome Res Rep 2024;3:36 https://dx.doi.org/10.20517/mrr.2024.06 Page 5 of 15

grade nitrogen source and applicable on a large scale. Moreover, as the growth rate on soy medium was
higher than that on mucin and relatively high cell densities were obtained, we decided to further
characterize A. muciniphila cells grown on this food-grade medium, the more so as these were highly active
[26]
in protecting mice from diet-induced obesity . A few observations were noted in the first series of
experiments that needed to be further addressed. First, phase-contrast and scanning electron microscopy of
cells grown on soy medium showed a significantly (P-value < 0.01) elongated shape with a length of 1.3
(±0.80) µm vs. 0.8 (±0.25) µm when grown in mucin medium (based on analysis of 251 and 160 cells,
respectively) [Supplementary Figure 1]. Additionally, the acetate/propionate ratio in the soy medium was
0.92, while that of cells grown in mucin was 1.2 (±0.13) . This can be explained by the fact that the
[32]
GlcNAc, which is present in equal amounts as glucose in the soy medium, generates an extra acetate after
deamination, as reported previously [24,25] .

To further address the global differences between A. muciniphila cells grown in soy medium and mucin
medium, transcriptome analysis was performed to reveal initial transcriptional changes between mucin and
soy medium (Supplementary Table 1 and see below).

The main differences were found to be in the increased expression in soy medium of genes encoding
transporters such as Major Facilitator Superfamily (MFS), biopolymer, anion and amino acid transporters
(Amuc_1331, Amuc_0546, Amuc_0221 and Amuc_0037), as well as peptide, aliphatic sulfonate, nitrate/
sulfonate/bicarbonate, cobalt and manganese ABC transporters (Amuc_0672, AMUC_1297, Amuc_0408,
Amuc_1198, Amuc_1199, Amuc_0056, Amuc_1380 and Amuc_1186) with an increase > 5-fold. In addition,
genes involved in oxygen stress response were found to be higher in soy medium including rubrerythrin
(Amuc_2055 and Amuc_2056), peroxidase (Amuc_1321), and catalase (Amuc_2070). In mucin medium,
genes involved in cell shape (Amuc_0540) and division (Amuc_0348) and mucin degradation genes such as
alpha-N-acetylglucosaminidase (Amuc_0060), beta-glucanase (Amuc_0875), and sulfatases (Amuc_0491
and Amuc_0451) were found to have an > 5-fold increase.

High biomass yield reached on food-grade medium
Because of the apparent effect of equimolar amounts of glucose and GlcNAc on the morphology, viscosity,
and gene expression of A. muciniphila cells, we further explored the effect of different carbon source ratios
on the growth and physiology of A. muciniphila. For this purpose, we decided to use pea peptone as an
additional food-grade nitrogen source rather than soy peptone to avoid potential issues associated with
phytoestrogens present in soy. A total of four fermentations were characterized in detail, with three different
glucose to GlcNAc carbon source ratios 3:1 (Condition A), 10:1 (Condition B), and 20:1 (Condition C) and
one control, which was supplemented with mucin (Condition D). Interestingly, while the mucin medium
enabled a rapid initiation of growth, an increase in the duration of the lag phase was observed along with
the decreasing concentrations of GlcNAc in the bioreactors [Figure 1]. It is important to note that the pre-
cultures were grown on pea peptone medium supplemented with equimolar concentrations of glucose and
GlcNAc, which is most similar to condition A in terms of glucose to GlcNAc ratio. However, up to four
transfers in food-grade medium supplemented with glucose and GlcNAc in a ratio of 20:1 were found to
lead to growth adaptation of A. muciniphila and rapid initiation of growth [Supplementary Figure 2].
Microscopy results showed the formation of elongated cells in conditions A-C as compared to mucin
(condition D) [Supplementary Figure 3]. The number of cells and increased cell length observed in these
cultures reflect a higher biomass production. Condition A was found to have the fastest growth and highest
biomass density, as deduced from the OD600 measurements.