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Diverse intestinal microbiota is frequently used in in vitro bioreactor models to study the effects of diet, chemical contaminations, or medication. However, the reproducible cultivation of fecal microbiota is challenging and the resultant communities behave highly dynamic. To approach the issue of reproducibility in in vitro models, we established an intestinal microbiota model community of reduced complexity, SIHUMIx, as a valuable model for in vitro use. The development of the SIHUMIx community was monitored over time with methods covering the cellular and the molecular level. We used microbial flow cytometry, intact protein profiling and terminal restriction fragment length polymorphism analysis to assess community structure. In parallel, we analyzed the functional level by targeted analysis of short-chain fatty acids and untargeted metabolomics. The stability properties constancy, resistance, and resilience were approached both on the structural and functional level of the community. We show that the SIHUMIx community is highly reproducible and constant since day 5 of cultivation. Furthermore, SIHUMIx has the ability to resist and recover from a pulsed perturbation, with changes in community structure recovered earlier than functional changes. Since community structure and function changed divergently, both levels need to be monitored at the same time to gain a full overview of the community development. All five methods are highly suitable to follow the community dynamics of SIHUMIx and indicated stability on day five. This makes SIHUMIx a suitable in vitro model to investigate the effects of e.g. medical, chemical, or dietary interventions.

Many functions in host-microbiota interactions are potentially influenced by intestinal transit times, but little is known about the effects of altered transition times on the composition and functionality of gut microbiota. To analyze these effects, we cultivated the model community SIHUMIx in bioreactors in order to determine the effects of varying transit times (TT) on the community structure and function. After five days of continuous cultivation, we investigated the influence of different medium TT of 12 h, 24 h, and 48 h. For profiling the microbial community, we applied flow cytometric fingerprinting and revealed changes in the community structure of SIHUMIx during the change of TT, which were not associated with changes in species abundances. For pinpointing metabolic alterations, we applied metaproteomics and metabolomics and found, along with shortening the TT, a slight decrease in glycan biosynthesis, carbohydrate, and amino acid metabolism and, furthermore, a reduction in butyrate, methyl butyrate, isobutyrate, valerate, and isovalerate concentrations. Specifically, B. thetaiotaomicron was identified to be affected in terms of butyrate metabolism. However, communities could recover to the original state afterward. This study shows that SIHUMIx showed high structural stability when TT changed-even four-fold. Resistance values remained high, which suggests that TTs did not interfere with the structure of the community to a certain degree.

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