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Huang et al. Microbiome Res Rep 2024;3:30 https://dx.doi.org/10.20517/mrr.2024.14 Page 7 of 13

Figure 3. Metabolomics revealed specific metabolite changes attributed to the host, continuous flow, and F. prausnitzii A2-165. (A) The
volcano plot of GuMI-NB vs. Static. P < 0.05, |fold change| > 2. Blue is significantly higher in static condition (Static) culture; red is
significantly higher in GuMI-NB; (B) Metabolites mapped to glycolysis and Krebs cycle pathways. Metabolites highlighted in red, blue,
and black are significantly higher, significantly lower, and not significantly changed in GuMI-NB vs. Static, respectively. Significance
threshold: P < 0.05, |fold change| > 2. Metabolites in grey are not detected; (C) The volcano plot of metabolite changes in GuMI-FP vs.
GuMI-NB. P < 0.05, |fold change| > 1.5. The rationale for lowering the threshold is that the continuous flow dilutes the produced
metabolites. Therefore, a 50% change in the chemical concentration is considered significant; One-way ANOVA was performed for
each compound in (B-D) to compare the differences between Static, GuMI-NB, and GuMI-FP. Note that the peak area ratio can not be
used to compare the levels of different compounds; (E) Nucleoside hydrolase gene in the genome of F. prausnitzii A2-165. Gene ID:
CG447_RS10420; accession ID of its predicted protein: C7H870 (UniProt). GuMI-NB: GuMI without bacteria; GuMI-FP: GuMI with
F. prausnitzii.

F. prausnitzii metabolizes host- and nutrient-derived nucleosides, nucleobases, and amino acids in
GuMI
In addition to lactate, F. prausnitzii can metabolize other compounds from the fresh medium or colonic
epithelium. For example, guanosine did not accumulate in the Static culture but was reliably detected in
GuMI-NB, indicating that it was mainly from the fresh medium and not from epithelial cells. However, the
level of guanosine in GuMI-FP significantly (P < 0.0001) dropped by ~20 times to a low level [Figure 3C].