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Page 4 of 15 Geerlings et al. Microbiome Res Rep 2024;3:36 https://dx.doi.org/10.20517/mrr.2024.06

(OrganoTechnie SAS, 32 g/L), and L-threonine (4 g/L). After autoclaving 2 mL of reducing solution
*
containing NaHCO (40 g/L) and L-cysteine HCl (5 g/L) and 1% (v/v) of vitamin solution (see above) were
3
added to the medium. The medium was inoculated with 1% (v/v) of the pre-culture. Anaerobic
fermentations using this medium were performed as described in the next section.

Anaerobic fermentation
The fermentations were conducted in four parallel bioreactors (DasGip, Eppendorf, Germany) using
700 mL of food-grade medium containing either glucose and GlcNAc in three different ratios (3:1, 10:1 or
20:1 glucose to GlcNAc, named A, B, and C, respectively) or mucus (Condition D) with a final total
concentration of 150 mM or 0.5% crude mucin. The pH was set at 6.8 and a stirring rate of 100 rpm was
applied with N /CO gas flow (80%/20%). The medium of all four bioreactors was inoculated with 1% (v/v)
2
2
of cultures pre-grown on tryptone medium. The fermentation was terminated after 72 h. Samples were
taken for optical density (OD) measurements, high performance liquid chromatography (HPLC) analysis,
microscopic analysis, RNA sequencing (triplicates), and proteomics (duplicates). Samples for HPLC
analysis were stored at -20 °C until use. Samples (10 mL) for RNA sequencing and proteomics were taken at
early, mid, and end exponential phases and centrifuged for 30 min at 4,700 rpm at 4 °C, after which the
supernatant was removed. Then, 1 mL of RNAlater was added to the pellets of the samples for RNA
sequencing. Lastly, the samples for both RNA sequencing (in triplicate) and proteomics (in duplicate) were
snap-frozen in liquid nitrogen and stored at -80 °C.

HPLC
Samples were obtained at different time points during the fermentation period for the analysis of
fermentation products. Crotonate was used as the internal standard. The external standards were GlcNAc,
glucose, acetate, propionate, succinate, lactate, and 1,2-propanediol. The substrates and fermentation
products were measured using a Shimadzu LC_2030C equipped with a refractive index detector and a
Shodex SH1011 column. Two runs were performed for each sample, employing oven temperatures of 45
and 75 °C, with pump flow rates of 1.0 and 0.9 mL/min, respectively. For both runs, 0.01N H SO was used
4
2
as eluent. All samples and standards (10 µL) ran for 15 min. The concentrations of the standards were
ranging between 2.5 and 60 mM. Lastly, the fermentation profiles obtained with HPLC were used to
calculate the carbon and energy balances at the endpoint of all fermentations.

RNA isolation and transcriptome analysis
[44]
RNA isolation was performed as described previously . Further processing of the total RNA was
performed by Novogene (Cambridge, United Kingdom) and paired-end sequences of 150 bp were obtained
using an Illumina platform. Transcriptome analysis has been performed as previously described . All
[33]
further analysis was done using R version 3.6.3 in Rstudio version 1.2.5019.

RESULTS
Growth characteristics and metabolic activity
In the first series of experiments, we built on the metabolic modeling data that predicted A. muciniphila
Muc to grow efficiently (growth rate of 0.13 h ) on an equimolar mixture of glucose and GlcNAc in a
-1
T
minimal medium with threonine [24,25] . To increase cell yield, a food-grade and plant-based protein source
was added to the minimal medium in the form of 16 g/L soy protein hydrolysate that resulted in a medium
(soy medium) yielding a high growth rate of 0.53 h exceeding that of A. muciniphila on mucin, which is
-1
approximately 0.41 h -1[12,35] . The cell densities in the soy peptone medium, as measured by absorption at
OD600, were above 5, whereas the mucin medium supported growth to an OD of 2-2.5 . Even higher cell
[24]
yields could be obtained by using pea peptone at a level of 32 g/L, which led to high densities of OD600
values above 10. Since the soy protein hydrolysate is derived from a plant source, it is an acceptable food-