Quantitative differences in synthetic gut microbial inoculums do not affect the final stabilized in vitro community compositions

In vitro studies of synthetic gut microbial communities (SGMCs) can provide valuable insights into the ecological structure and function of gut microbiota. However, the importance of the quantitative composition of an SGMC inoculum and its effect on the eventual stable in vitro microbial community has not been studied. To address this, we constructed two 114-member SGMCs differing only in their quantitative composition-one reflecting the average human fecal microbiome and another mixed in equal proportions based on cell counts. We inoculated each in an automated anaerobic multi-stage in vitro gut fermentor simulating two different colonic conditions, mimicking proximal and distal colons. We replicated this setup with two different nutrient media, periodically sampled the cultures for 27 days, and profiled their microbiome compositions using 16S rRNA gene amplicon sequencing. While nutrient medium explained 36% of the variance in microbiome composition, initial inoculum composition failed to show a statistically significant effect. Under all four conditions, paired fecal and equal SGMC inoculums converged to reach stable community compositions resembling each other. Our results have broad implications for simplifying in vitro SGMC investigations. IMPORTANCE In vitro cultivation of synthetic gut microbial communities (SGMCs) can provide valuable insights into the ecological structure and function of gut microbiota. However, it is currently not known whether the quantitative composition of the initial inoculum can influence the eventual stable in vitro community structure. Hence, using two SGMC inoculums consisting of 114 unique species mixed in either equal proportions (Eq inoculum) or resembling proportions in an average human fecal microbiome (Fec inoculum), we show that initial inoculum compositions did not influence the final stable community structure in a multi-stage in vitro gut fermentor. Under two different nutrient media and two different colon conditions (proximal and distal), both Fec and Eq communities converged to resemble each other's community structure. Our results suggest that the time-consuming preparation of SGMC inoculums may not be needed and has broad implications for in vitro SGMC studies.