Establishment of an In Vitro System of the Human Intestinal Microbiota: Effect of Cultivation Conditions and Influence of Three Donor Stool Samples

Abstract

Fecal microbiota transplantation (FMT) is an alternative method for the treatment of gastrointestinal diseases with a high recovery rate. Disadvantages are ethical concerns, high donor requirements and the low storability of stool samples. The cultivation of an in vitro microbiota in a continuous bioreactor was established as an alternative to FMT to overcome these problems. In this study, the influence of the system parameters and donor stool characteristics was investigated. Each continuous colonic fermentation system was inoculated with feces from three different donors until a stable state was established. The influence of the fermentation conditions on the system’s behavior regarding cell count, metabolic activity, short-chain fatty acid profile and microbiota composition as well as richness and diversity was assessed. Cultivation conditions were found to affect the microbial system: the number of cells and the production of short-chain fatty acids increased. The abundance of Actinobacteria and Firmicutes decreased, Bacteroidetes increased, while Proteobacteria and Verrucomicrobia remained largely unaffected. Diversity in the in vitro system decreased, but richness was unaffected. The cultivation of stool from different donors revealed that the performance of the created in vitro system was similar and comparable, but unique characteristics of the composition of the original stool remained.

Influence of Cultivation pH on Composition, Diversity, and Metabolic Production in an In Vitro Human Intestinal Microbiota

“…For donor B, the stable system was reached after 75.9 ± 1.0 h (pH 6.0), and after 74.1 ± 0.2 h (pH 6.5) for donor C. Consequently, a stable system can be reached for each pH value and system after 77 h the latest. These results are in accordance with former studies, where the system used here was established [38]. When comparing systems, only values in the stable system after 77-120 h of processing time were considered.…”

“…Nevertheless, these results are comparable with former studies [38] as well as other in vitro studies [29]. Bircher et al detected similar concentrations for acetate and butyrate in their in vitro microbiome.…”

“…For the cultivation of the human intestinal microbiota, a stable in vitro system was used [38]. The aim of this study was to reveal the influence of the pH value on the cultivation outcome in the stable system.…”

“…Continuous flow fermentation medium (CFF) was used for the preparation of the inoculum as well as in the cultivation system itself. The composition and production of CFF medium were already described in previous studies [38]. After preparation, the final medium was stored at + 4 • C and used within 72 h for either preparation of the inoculum or the medium for the cultivation system.…”

“…These conditions reflect the mean retention time in the human colon [37]. In a previous study, the establishment of a simplified system as well as the influence of different donor stool characteristics were investigated [38]. For gas supply conditions and cultivation temperature, all existing systems suggest similar parameters.…”