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Page 6 of 13 Huang et al. Microbiome Res Rep 2024;3:30 https://dx.doi.org/10.20517/mrr.2024.14

Figure 2. Metabolite profile in the apical compartment in static condition (Static) and GuMI culture with and without bacteria. (A) The
principal component analysis plot indicates a clear separation of the analyzed metabolome among the three groups. In total, 76 detected
compounds were included; (B) A distance plot of all the samples shows the inter-batch variation. In total, four batches of GuMI
experiments were carried out. The samples are from four experiments as follows: experiment 1: Static_1 and 2, GuMI-NB_1, GuMI-FP_1
and 2; experiment 2: Static_3, 4, and 5, GuMI-NB_2 and 3, GuMI-FP_3 and 4; experiment 3: Static_6, GuMI-NB_4 and 5, GuMI-FP_5 and
6; experiment 4: Static_7 and 8, GuMI-NB_6, 7, and 8, GuMI-FP_7 and 8; (C) Heatmap of detected metabolites in Static, GuMI-NB, and
GuMI-FP. Clusters 1, 2, 3, and 4 indicate the metabolites accumulated in specific conditions. GuMI-NB: GuMI without bacteria; GuMI-FP:
GuMI with F. prausnitzii.

respectively [Figure 2C]. For example, chemicals in cluster 1 were found to be at the highest level in
GuMI-FP, suggesting that F. prausnitzii produces these metabolites. Additionally, metabolites in cluster 3
were at the highest level in Static culture, suggesting that these metabolites are produced by colonic
epithelium and washed away by the flow in GuMI [Figure 2C]. Together, these results indicate that the
GuMI coculture system, in combination with metabolomics, can effectively discern the influence of
F. prausnitzii, epithelial cells, and flow on the metabolites in the apical compartment.

Colonic epithelium consumes amino acids and secretes diverse metabolites related to the
glycolysis pathway
Next, we sought to determine what metabolites were specifically consumed or secreted by colonic epithelial
cells. Twelve metabolites were significantly higher in GuMI-NB than in Static (P < 0.05, fold change > 2,
Figure 3A), and these metabolites belong to amino acids, nucleosides, and nucleobases. These results
suggest that these compounds are primarily from the apical medium and consumed by colonic epithelial
cells. Specifically, we found glutamate consumption is accompanied by the production of its metabolite
alpha-ketoglutarate, which is accumulated in Static [Figure 3A]. Alpha-ketoglutarate can also be generated
by glycolysis and Krebs cycle. Correspondingly, we found that pyruvate, lactate, citrate, alpha-ketoglutarate,
and malate were significantly accumulated in Static culture [Figure 3B]. Other glycolysis and Krebs
pathways metabolites were also detected but did not change significantly [Figure 3B]. Together, these results
suggest that continuous flow in GuMI supplies amino acids and nucleosides for colonic epithelial cells and
washes away many metabolites produced by colonic epithelial cell metabolism.